Gip receptor-active glucagon compounds

ABSTRACT

Glucagon peptides with increased GIP activity are provided, optionally with GLP-I and/or glucagon activity. In some embodiments, C-terminally extended glucagon peptides comprising an amino acid sequence substantially similar to native glucagon are provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/187,578 filed Jun. 16, 2009, theentirety of which is hereby incorporated by reference herein.

BACKGROUND

Pre-proglucagon is a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofpre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide thatcorresponds to amino acids 72 through 108 of pre-proglucagon. GLP-1(7-36) amide or GLP-1 (7-37) acid are biologically potent forms ofGLP-1, that demonstrate essentially equivalent activity at the GLP-1receptor.

Hypoglycemia occurs when blood glucose levels drops too low to provideenough energy for the body's activities. In adults or children olderthan 10 years, hypoglycemia is uncommon except as a side effect ofdiabetes treatment, but it can result from other medications ordiseases, hormone or enzyme deficiencies, or tumors. When blood glucosebegins to fall, glucagon, a hormone produced by the pancreas, signalsthe liver to break down glycogen and release glucose, causing bloodglucose levels to rise toward a normal level. Thus, glucagon's mostrecognized role in glucose regulation is to counteract the action ofinsulin and maintain blood glucose levels. However for diabetics, thisglucagon response to hypoglycemia may be impaired, making it harder forglucose levels to return to the normal range.

Hypoglycemia is a life threatening event that requires immediate medicalattention. The administration of glucagon is an established medicationfor treating acute hypoglycemia and it can restore normal levels ofglucose within minutes of administration. When glucagon is used in theacute medical treatment of hypoglycemia, a crystalline form of glucagonis solubilized with a dilute acid buffer and the solution is injectedintramuscularly. While this treatment is effective, the methodology iscumbersome and dangerous for someone that is semi-conscious.Accordingly, there is a need for a glucagon analog that maintains orexceeds the biological performance of the parent molecule but issufficiently soluble and stable, under relevant physiologicalconditions, that it can be pre-formulated as a solution, ready forinjection.

Additionally, diabetics are encouraged to maintain near normal bloodglucose levels to delay or prevent microvascular complications.Achievement of this goal usually requires intensive insulin therapy. Instriving to achieve this goal, physicians have encountered a substantialincrease in the frequency and severity of hypoglycemia in their diabeticpatients. Accordingly, improved pharmaceuticals and methodologies areneeded for treating diabetes that are less likely to induce hypoglycemiathan current insulin therapies.

GLP-1 has different biological activities compared to glucagon. Itsactions include stimulation of insulin synthesis and secretion,inhibition of glucagon secretion, and inhibition of food intake. GLP-1has been shown to reduce hyperglycemia (elevated glucose levels) indiabetics. Exendin-4, a peptide from lizard venom that shares about 50%amino acid identity with GLP-1, activates the GLP-1 receptor andlikewise has been shown to reduce hyperglycemia in diabetics.

There is also evidence that GLP-1 and exendin-4 may reduce food intakeand promote weight loss, an effect that would be beneficial not only fordiabetics but also for patients suffering from obesity. Patients withobesity have a higher risk of diabetes, hypertension, hyperlipidemia,cardiovascular disease, and musculoskeletal diseases.

Accordingly, there remains a need for alternative and preferablyimproved methods for treating diabetes and obesity.

SUMMARY

As described herein, analogs of glucagon peptides which exhibit agonistactivity at the GIP receptor are provided by the invention. Methods ofusing such analogs are additionally provided herein.

Native glucagon (SEQ ID NO: 1) does not activate the GIP receptor andtypically, native glucagon exhibits essentially 0% (e.g., less than0.001%, less than 0.0001%) activity of native GIP at the GIP receptor.Native glucagon also has about 1% of the activity of native-GLP-1 at theGLP-1 receptor. Modifications to the native glucagon sequence (SEQ IDNO: 1) are described herein which produce analogs of glucagon peptidesthat can exhibit potent glucagon activity equivalent to or better thanthe activity of native glucagon (SEQ ID NO: 1), potent GIP activityequivalent to or better than the activity of native GIP (SEQ ID NO:675), and/or potent GLP-1 activity equivalent to or better than theactivity of native GLP-1. GLP-1 (7-36) amide (SEQ ID NO: 52) or GLP-1(7-37) (acid) (SEQ ID NO: 50) are biologically potent forms of GLP-1,that demonstrate essentially equivalent activity at the GLP-1 receptor.

The data described herein show that analogs of glucagon having both GIPactivity and GLP-1 activity are particularly advantageous for inducingweight loss or preventing weight gain, as well as for treatinghyperglycemia, including diabetes. This activity is particularlyunexpected in view of teachings in the art that antagonizing GIP isdesirable for reducing daily food intake and body weight, and increasinginsulin sensitivity and energy expenditure. (Irwin et al., Diabetologia50: 1532-1540 (2007); and Althage et al., J Biol Chem, e-publication onApr. 17, 2008).

Accordingly, in some embodiments, the analogs of glucagon peptidesdescribed herein exhibit an EC50 for GIP receptor activation activity ofabout 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4, 3, 2 or 1 nMor less. In some embodiments, the analogs described herein exhibit anEC50 at the GIP receptor that is about 0.001 nM, 0.01 nM, or 0.1 nM. Insome embodiments, the analogs described herein exhibit an EC50 at theGIP receptor that is no more than about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 75 nM, or 100nM. In some embodiments, the analogs exhibit an EC50 for glucagonreceptor activation of about 100 nM or less, or about 75, 50, 25, 10, 8,6, 5, 4, 3, 2 or 1 nM or less. In some embodiments, the analogsdescribed herein exhibit an EC50 at the glucagon receptor that is about0.001 nM, 0.01 nM, or 0.1 nM. In some embodiments, the EC50 at theglucagon receptor is no more than about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6nM, 8 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 75 nM, or 100nM. In some embodiments, the analogs exhibit an EC50 for GLP-1 receptoractivation of about 100 nM or less, or about 75, 50, 25, 10, 8, 6, 5, 4,3, 2 or 1 nM or less. In some embodiments, the analogs described hereinexhibit an EC50 at the GLP-1 receptor that is about 0.001 nM, 0.01 nM,or 0.1 nM. In some embodiments, the EC50 at the GLP-1 receptor is nomore than about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 8 nM, 10 nM, 15 nM,20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 75 nM, or 100 nM. Receptor activationcan be measured by in vitro assays measuring cAMP induction in HEK293cells over-expressing the receptor, e.g. assaying HEK293 cellsco-transfected with DNA encoding the receptor and a luciferase genelinked to cAMP responsive element as described in Example 14.

In some embodiments, the analogs exhibit at least about 0.005%, 0.0075%,0.01%, 0.025%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%,175% or 200% or higher activity at the GIP receptor relative to nativeGIP (GIP potency). In some embodiments, the analogs described hereinexhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity atthe GIP receptor relative to native GIP. In some embodiments, theanalogs exhibit at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%or higher activity at the glucagon receptor relative to native glucagon(glucagon potency). In some embodiments, the analogs described hereinexhibit no more than 1000%, 10,000%, 100,000%, or 1,000,000% activity atthe glucagon receptor relative to native glucagon. In some embodiments,the analogs exhibit at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 75%, 100%, 125%, 150%, 175% or 200% or higher activity atthe GLP-1 receptor relative to native GLP-1 (GLP-1 potency). In someembodiments, the analogs described herein exhibit no more than 1000%,10,000%, 100,000%, or 1,000,000% activity at the GLP-1 receptor relativeto native GLP-1.

In certain embodiments, the analogs and glucagon peptides describedherein exhibit the indicated % activity, when lacking a hydrophilicmoiety, e.g., PEG, but exhibit a decreased % activity (e.g., about a10-fold decrease in activity), when comprising a hydrophilic moiety,e.g., PEG. Accordingly, in some embodiments, the analogs exhibit theaforementioned % activity levels when lacking a hydrophilic moiety andexhibit about a 10-fold decrease in activity when comprising ahydrophilic moiety. An analog's activity at a receptor relative to anative ligand of the receptor is calculated as the inverse ratio ofEC50s for the analog vs. the native ligand.

Thus, one aspect of the invention provides analogs that exhibit activityat both the glucagon receptor and the GIP receptor (“glucagon/GIPco-agonists”). These analogs have lost native glucagon's selectivity forglucagon receptor compared to GIP receptor. In some embodiments, theEC50 of the analog at the GIP receptor is less than about 50-fold,40-fold, 30-fold or 20-fold different (higher or lower) from its EC50 atthe glucagon receptor. In some embodiments, the GIP potency of theanalog is less than about 500-, 450-, 400-, 350-, 300-, 250-, 200-,150-, 100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher orlower) from its glucagon potency. In some embodiments, the ratio of theEC50 of the analog at the GIP receptor divided by the EC50 of the analogat the glucagon receptor is less than about 100, 75, 60, 50, 40, 30, 20,15, 10, or 5. In some embodiments, the ratio of the EC50 at the GIPreceptor divided by the EC50 at the glucagon receptor is about 1 or lessthan about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05,0.067, 0.1, 0.2). In some embodiments, the ratio of the GIP potency ofthe analog compared to the glucagon potency of the analog is less thanabout 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 60, 50, 40, 30,20, 15, 10, or 5. In some embodiments, the ratio of the potency at theGIP receptor divided by the potency at the glucagon receptor is about 1or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025,0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, GLP-1 activity hasbeen significantly reduced or destroyed, e.g., by an amino acidmodification at position 7, e.g., substitution with Ile, a deletion ofthe amino acid(s) C-terminal to the amino acid at position 27 or 28,yielding a 27- or 28-amino acid peptide, or a combination thereof.

Another aspect of the invention provides analogs that exhibit activityat the glucagon, GIP and GLP-1 receptors (“glucagon/GIP/GLP-1tri-agonists”). These analogs have lost native glucagon's selectivityfor the glucagon receptor compared to both the GLP-1 and GIP receptors.In some embodiments, the EC50 of the analog at the GIP receptor is lessthan about 5000-fold, 2500-fold, 1000-fold, 750-fold, 500-fold,250-fold, 100-fold, 50-fold, 40-fold, 30-fold or 20-fold different(higher or lower) from its respective EC50s at the glucagon and GLP-1receptors. In some embodiments, the GIP potency of the analog is lessthan about 1000-, 750-, 500-, 450-, 400-, 350-, 300-, 250-, 200-, 150-,100-, 75-, 50-, 25-, 20-, 15-, 10-, or 5-fold different (higher orlower) from its glucagon and GLP-1 potencies. In some embodiments, theratio of the EC50 of the tri-agonist at the GIP receptor divided by theEC50 of the tri-agonist at the GLP-1 receptor is less than about 10,000,7500, 5000, 2500, 1000, 750, 500, 250, 100, 75, 60, 50, 40, 30, 20, 15,10, 5, or 1. In some embodiments, the ratio of the EC50 at the GLP-1receptor divided by the EC50 at the GIP receptor is about 5, 4, 3, 2, or1 or less than about 1 (e.g., less than about 0.00001, 0.0001, 0.001,0.0025, 0.005, 0.0075, 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05,0.067, 0.1, 0.2). In some embodiments, the EC50 at the GLP-1 receptor isgreater than about 0.1 (e.g., greater than about 0.25, greater thanabout 0.5, greater than about 0.75, greater than about 1). In someembodiments, the ratio of the GIP potency of the tri-agonist compared tothe GLP-1 potency of the tri-agonist is less than about 1000, 750, 500,250, 100, 75, 60, 50, 40, 30, 20, 15, 10, 5, or 1. In some embodiments,the ratio of the potency at the GIP receptor divided by the potency atthe GLP-1 receptor is about 5, 4, 3, 2, or 1, or less than about 1(e.g., less than about 0.0001, 0.001, 0.01, 0.013, 0.0167, 0.02, 0.025,0.03, 0.05, 0.067, 0.1, 0.2). In related embodiments, the ratio of theEC50 of the tri-agonist at the GIP receptor divided by the EC50 of thetri-agonist at the glucagon receptor is less than about 100, 75, 60, 50,40, 30, 20, 15, 10, or 5. In some embodiments, the ratio of the EC50 atthe GIP receptor divided by the EC50 at the glucagon receptor is about 1or less than about 1 (e.g., about 0.01, 0.013, 0.0167, 0.02, 0.025,0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIPpotency of the tri-agonist compared to the glucagon potency of thetri-agonist is less than about 500, 450, 400, 350, 300, 250, 200, 150,100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, theratio of the potency at the GIP receptor divided by the potency at theglucagon receptor is about 1 or less than about 1 (e.g., about 0.01,0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). In someembodiments, the ratio of the EC50 of the tri-agonist at the GLP-1receptor divided by the EC50 of the tri-agonist at the glucagon receptoris less than about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In someembodiments, the ratio of the EC50 at the GLP-1 receptor divided by theEC50 at the glucagon receptor is about 1 or less than about 1 (e.g.,about 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2). Insome embodiments, the ratio of the GLP-1 potency of the tri-agonistcompared to the glucagon potency of the tri-agonist is less than about100, 75, 60, 50, 40, 30, 20, 15, 10, or 5. In some embodiments, theratio of the potency at the GLP-1 receptor divided by the potency at theglucagon receptor is about 1 or less than about 1 (e.g., about 0.01,0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1, 0.2).

Yet another aspect of the invention provides analogs that exhibitactivity at the GLP-1 and GIP receptors, but in which the glucagonactivity has been significantly reduced or destroyed (“GIP/GLP-1co-agonists”), e.g., by an amino acid modification at position 3. Forexample, substitution at this position with an acidic, basic, or ahydrophobic amino acid (glutamic acid, ornithine, norleucine) reducesglucagon activity. In some embodiments, the EC50 of the analog at theGIP receptor is less than about 1000-fold, 750-fold, 500-fold, 250-fold,100-fold, 50-fold, 40-fold, 30-fold or 20-fold different (higher orlower) from its EC50 at the GLP-1 receptor. In some embodiments, the GIPpotency of the analog is less than about 1000-, 750-, 500-, 250-, 100-,25-, 20-, 15-, 10-, or 5-fold different (higher or lower) from its GLP-1potency. In some embodiments these analogs have about 10% or less of theactivity of native glucagon at the glucagon receptor, e.g. about 1-10%,or about 0.1-10%, or greater than about 0.1% but less than about 10%. Insome embodiments, the ratio of the EC50 of the analog at the GIPreceptor divided by the EC50 of the analog at the GLP-1 receptor is lessthan about 100, 75, 60, 50, 40, 30, 20, 15, 10, or 5, and no lessthan 1. In some embodiments, the ratio of the GIP potency of the analogcompared to the GLP-1 potency of the analog is less than about 100, 75,60, 50, 40, 30, 20, 15, 10, or 5, and no less than 1. In someembodiments, the ratio of the EC50 of the co-agonist at the GIP receptordivided by the EC50 of the co-agonist at the GLP-1 receptor is less thanabout 10,000, 7500, 5000, 2500, 1000, 750, 500, 250, 100, 75, 60, 50,40, 30, 20, 15, 10, 5, or 1. In some embodiments, the ratio of the EC50at the GLP-1 receptor divided by the EC50 at the GIP receptor is about5, 4, 3, 2, or 1 or less than about 1 (e.g., less than about 0.00001,0.0001, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.013, 0.0167, 0.02, 0.025,0.03, 0.05, 0.067, 0.1, 0.2). In some embodiments, the ratio of the GIPpotency of the co-agonist compared to the GLP-1 potency of theco-agonist is less than about 1000, 750, 500, 250, 100, 75, 60, 50, 40,30, 20, 15, 10, 5, or 1. In some embodiments, the ratio of the potencyat the GIP receptor divided by the potency at the GLP-1 receptor isabout 5, 4, 3, 2, or 1, or less than about 1 (e.g., less than about0.0001, 0.001, 0.01, 0.013, 0.0167, 0.02, 0.025, 0.03, 0.05, 0.067, 0.1,0.2).

A further aspect of the invention provides analogs that exhibit activityat the GIP receptor, in which the glucagon and GLP-1 activity have beensignificantly reduced or destroyed (“GIP agonist glucagon peptides”),e.g., by amino acid modifications at positions 3 and 7. In someembodiments these analogs have about 10% or less of the activity ofnative glucagon at the glucagon receptor, e.g. about 1-10%, or about0.1-10%, or greater than about 0.1%, 0.5%, or 1% but less than about 1%,5%, or 10%. In some embodiments these analogs also have about 10% orless of the activity of native GLP-1 at the GLP-1 receptor, e.g. about1-10%, or about 0.1-10%, or greater than about 0.1%, 0.5%, or 1% butless than about 1%, 5%, or 10%.

In some aspects of the invention, the analogs which exhibit agonistactivity at the GIP receptor comprise SEQ ID NO: 1 with at least oneamino acid modification and an extension of 1 to 21 amino acids (e.g., 5to 18, 7 to 15, 9 to 12 amino acids) C-terminal to the amino acid atposition 29 of the analog.

In certain aspects, the analogs comprise at least one amino acidmodification and up to 15 amino acid modifications (e.g., no more than15 amino acid modifications, no more than 10 amino acid modifications).For example, the analog can comprise SEQ ID NO: 1 with 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid modifications. In certainembodiments, the analogs comprise at least one amino acid modificationat up to 10 amino acid modifications and additional conservative aminoacid modifications. In further aspects, at least one of the amino acidmodifications confers a stabilized alpha helix structure in theC-terminal portion of the analog. Modifications which achieve astabilized alpha helix structure are described herein. See, for example,the teachings under the section entitled Stabilization of the alphahelix/Intramolecular bridges.

Analogs comprising an acylated or alkylated C-terminal extension or aC-terminal extension comprising 1-6 positive-charged amino acidsunexpectedly exhibited an increased agonist activity at the GIPreceptor. Thus, in certain aspects, at least one of the amino acids ofthe extension located at any of positions 37-43 (according to thenumbering of SEQ ID NO: 1) comprises an acyl or alkyl group which isnon-native to a naturally-occurring amino acid, i.e., the extension isacylated or alkylated. In some embodiments, the acyl or alkyl group isattached directly to the amino acid, e.g., via the side chain of theamino acid. In other embodiments, the acyl or alkyl group is attached tothe amino acid via a spacer (e.g., an amino acid, a dipeptide, atripeptide, a hydrophilic bifunctional spacer, a hydrophobicbifunctional spacer). Suitable amino acids comprising an acyl or alkylgroup, as well as suitable acyl groups and alkyl groups, are describedherein. See, for example, the teachings under the sections entitledAcylation and Alkylation.

In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 aminoacids) of the extension are positive-charged amino acids, e.g., aminoacids of Formula IV, such as, for example, Lys. As used herein, the term“positive-charged amino acid” refers to any amino acid,naturally-occurring or non-naturally occurring, comprising a positivecharge on an atom of its side chain at a physiological pH (e.g., pH 6.8to 8.0, pH 7.0 to 7.7). In certain aspects, the positive-charged aminoacids are located at any of positions 37, 38, 39, 40, 41, 42, and 43. Inspecific embodiments, a positive-charged amino acid is located atposition 40.

In other instances, the extension is acylated or alkylated as describedherein and comprises 1-6 positive charged amino acids as describedherein.

In yet other embodiments, the analogs which exhibit agonist activity atthe GIP receptor comprises (i) SEQ ID NO: 1 with at least one amino acidmodification, (ii) an extension of 1 to 21 amino acids (e.g., 5 to 18, 7to 15, 9 to 12 amino acids) C-terminal to the amino acid at position 29of the analog, and (iii) an amino acid comprising an acyl or alkyl groupwhich is non-native to a naturally-occurring amino acid which is locatedoutside of the C-terminal extension (e.g., at any of positions 1-29). Insome embodiments, the analog comprises an acylated or alkylated aminoacid at position 10. In particular aspects, the acyl or alkyl group is aC4 to C30 fatty acyl or C₄ to C₃₀ alkyl group. In some embodiments, theacyl or alkyl group is attached via a spacer, e.g., an amino acid,dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobicbifunctional spacer). In certain aspects, the analog comprises an aminoacid modification which stabilizes the alpha helix, such as a saltbridge between a Glu at position 16 and a Lys at position 20, or analpha,alpha-disubstituted amino acid at any one, two, three, or more ofpositions 16, 20, 21, and 24. In specific aspects, the analogadditionally comprises amino acid modifications which confer DPP-IVprotease resistance. Analogs comprising further amino acid modificationsare contemplated herein.

In certain embodiments, the analogs having GIP receptor activity exhibitat least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%)activity of native GIP at the GIP receptor. In some embodiments, theanalogs exhibit more than 20% (e.g., more than 50%, more than 75%, morethan 100%, more than 200%, more than 300%, more than 500%) activity ofnative GIP at the GIP receptor. In some embodiments, the analog exhibitsappreciable agonist activity at one or both of the GLP-1 and glucagonreceptors. In some aspects, the selectivity for these receptors (GIPreceptor and GLP-1 receptor and/or glucagon receptor) are within100-fold. For example, the selectivity for the GLP-1 receptor of theanalogs having GIP receptor activity can be less than 100-fold, within50-fold, within 25 fold, within 15 fold, within 10 fold) the selectivityfor the GIP receptor and/or the glucagon receptor.

As described herein, high potency glucagon agonist analogs are providedthat also exhibit increased activity at the glucagon receptor, and infurther embodiments exhibit enhanced biophysical stability and/oraqueous solubility. In addition, in accordance with another aspect ofthe invention, glucagon agonist analogs are provided that have lostnative glucagon's selectivity for the glucagon receptor verses the GLP-1receptor, and thus represent co-agonists of those two receptors.Selected amino acid modifications within the glucagon analogs cancontrol the relative activity of the analog at the GLP-1 receptor versesthe glucagon receptor. Thus, yet another aspect of the inventionprovides glucagon co-agonist analogs that have higher activity at theglucagon receptor versus the GLP-1 receptor, glucagon co-agonist analogsthat have approximately equivalent activity at both receptors, andglucagon co-agonist analogs that have higher activity at the GLP-1receptor versus the glucagon receptor. The latter category of co-agonistcan be engineered to exhibit little or no activity at the glucagonreceptor, and yet retain ability to activate the GLP-1 receptor with thesame or better potency than native GLP-1. Any of these analogs may alsoinclude modifications that confer enhanced biophysical stability and/oraqueous solubility.

Glucagon analogs that demonstrate co-agonism at the glucagon and GLP-1receptors are advantageous for several applications. First of all theuse of glucagon to treat hypoglycemia may overcompensate for low bloodglucose levels and result in excess blood glucose levels. If aglucagon/GLP-1 receptor co-agonist is administered, the additional GLP-1stimulation may buffer the glucagon agonist effect to prevent excessiveglucose blood levels resulting from treatment of hypoglycemia.

In addition as described herein, glucagon co-agonist analogs of theinvention may be used to control hyperglycemia, or to induce weight lossor prevent weight gain, when administered alone or in combination withother anti-diabetic or anti-obesity treatments. Another compound thatinduces weight loss is oxyntomodulin, a naturally occurring digestivehormone found in the small intestine (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e., SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 27 (KRNRNNIA). While thepresent invention contemplates that glucagon analogs described hereinmay optionally be joined to this 8 amino acid carboxy terminal extension(SEQ ID NO: 27), the invention in some embodiments also specificallycontemplates analogs and uses of analogs lacking the 8 contiguouscarboxy amino acids of SEQ ID NO: 27.

The compounds can be customized by amino acid modifications to regulatethe GLP-1 activity of the peptide, and thus the glucagon analogs of thepresent can be tailored to treat a particular condition or disease. Moreparticularly, glucagon analogs are provided herein wherein each analogdisplays a characteristic relative level of activity at the respectiveglucagon and GLP-1 receptors. For example, modifications can be made toeach peptide to produce a glucagon peptide having anywhere from at leastabout 1% (including at least about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%,50%, 60%, 75%, 100%, 125%, 150%, 175%) to about 200% or higher activityat the GLP-1 receptor relative to native GLP-1 and anywhere from atleast about 1% (including about 1.5%, 2%, 5%, 7%, 10%, 20%, 30%, 40%,50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%,450%) to about 500% or higher activity at the glucagon receptor relativeto native glucagon. In some embodiments, the glucagon peptides describedherein exhibit no more than about 100%, 1000%, 10,000%, 100,000%, or1,000,000% of the activity of native glucagon at the glucagon receptor.In some embodiments, the glucagon peptides described herein exhibit nomore than about 100%, 1000%, 10,000%, 100,000%, or 1,000,000% of theactivity of native GLP-1 at the GLP-1 receptor. The amino acid sequenceof native glucagon is SEQ ID NO: 1, the amino acid sequence of GLP-1(7-36)amide is SEQ ID NO: 52, and the amino acid sequence of GLP-1(7-37)acid is SEQ ID NO: 50. In exemplary embodiments, a glucagonpeptide may exhibit at least 10% of the activity of native glucagon atthe glucagon receptor and at least 50% of the activity of native GLP-1at the GLP-1 receptor, or at least 40% of the activity of nativeglucagon at the glucagon receptor and at least 40% of the activity ofnative GLP-1 at the GLP-1 receptor, or at least 60% of the activity ofnative glucagon at the glucagon receptor and at least 60% of theactivity of native GLP-1 at the GLP-1 receptor.

Selectivity of a glucagon peptide for the glucagon receptor versus theGLP-1 receptor can be described as the relative ratio of glucagon/GLP-1activity (the peptide's activity at the glucagon receptor relative tonative glucagon, divided by the peptide's activity at the GLP-1 receptorrelative to native GLP-1). For example, a glucagon peptide that exhibits60% of the activity of native glucagon at the glucagon receptor and 60%of the activity of native GLP-1 at the GLP-1 receptor has a 1:1 ratio ofglucagon/GLP-1 activity. Exemplary ratios of glucagon/GLP-1 activityinclude about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or10:1, or about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.5.As an example, a glucagon/GLP-1 activity ratio of 10:1 indicates a10-fold selectivity for the glucagon receptor versus the GLP-1 receptor.Similarly, a GLP-1/glucagon activity ratio of 10:1 indicates a 10-foldselectivity for the GLP-1 receptor versus the glucagon receptor.

In accordance with one embodiment, analogs of glucagon are provided thathave enhanced potency and optionally improved solubility and stability.In one embodiment, enhanced glucagon potency is provided by an aminoacid modification at position 16 of native glucagon (SEQ ID NO: 1). Byway of nonlimiting example, such enhanced potency can be provided bysubstituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms. In one embodimentthe enhanced potency glucagon agonist comprises a peptide of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7or a glucagon agonist analog of SEQ ID NO: 5. In accordance with oneembodiment a glucagon analog protein having enhanced potency at theglucagon receptor relative to wild type glucagon is provided wherein thepeptide comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10, wherein the glucagon peptide retains its selectivityfor the glucagon receptor relative to the GLP-1 receptors.

Glucagon receptor activity can be reduced, maintained, or enhanced by anamino acid modification at position 3, e.g. substitution of thenaturally occurring glutamine at position 3. In one embodiment,substitution of the amino acid at position 3 with an acidic, basic, orhydrophobic amino acid (glutamic acid, ornithine, norleucine) has beenshown to substantially reduce or destroy glucagon receptor activity. Theanalogs that are substituted with, for example, glutamic acid,ornithine, or norleucine have about 10% or less of the activity ofnative glucagon at the glucagon receptor, e.g. about 1-10%, or about0.1-10%, or greater than about 0.1% but less than about 10%, whileexhibiting at least 20% of the activity of GLP-1 at the GLP-1 receptor.For example, exemplary analogs described herein have about 0.5%, about1% or about 7% of the activity of native glucagon, while exhibiting atleast 20% of the activity of GLP-1 at the GLP-1 receptor.

In another embodiment, the naturally occurring glutamine at position 3of the glucagon peptide can be substituted with a glutamine analogwithout a substantial loss of activity at the glucagon receptor, and insome cases, with an enhancement of glucagon receptor activity. Forexample, a glucagon peptide comprising a glutamine analog at position 3may exhibit about 5%, about 10%, about 20%, about 50%, or about 85% orgreater the activity of native glucagon (e.g. SEQ ID NO: 1) at theglucagon receptor. In some embodiments a glucagon peptide comprising aglutamine analog at position 3 may exhibit about 20%, about 50%, about75%, about 100%, about 200% or about 500% or greater the activity of acorresponding glucagon peptide having the same amino acid sequence asthe peptide comprising the glutamine analog, except for the modifiedamino acid at position 3 (e.g. SEQ ID NO: 601 or SEQ ID NO: 602) at theglucagon receptor. In some embodiments, a glucagon peptide comprising aglutamine analog at position 3 exhibits enhanced activity at theglucagon receptor, but the enhanced activity is no more than 1000%,10,000%, 100,000%, or 1,000,000% of the activity of native glucagon orof a corresponding glucagon peptide having the same amino acid sequenceas the peptide comprising the glutamine analog, except for the modifiedamino acid at position 3.

In some embodiments, the glutamine analog is a naturally occurring or anon-naturally occurring amino acid comprising a side chain of StructureI, II or III:

wherein R¹ is C₀₋₃ alkyl or C₀₋₃ heteroalkyl; R² is NHR⁴ or C₁₋₃ alkyl;R³ is C₁₋₃ alkyl; R⁴ is H or C₁₋₃ alkyl; X is NH, O, or S; and Y isNHR⁴, SR³, or OR³. In some embodiments, X is NH or Y is NHR⁴. In someembodiments, R¹ is C₀₋₂ alkyl or C₁ heteroalkyl. In some embodiments, R²is NHR⁴ or C₁ alkyl. In some embodiments, R⁴ is H or C¹ alkyl. Inexemplary embodiments of Structure I, R¹ is CH₂—S, X is NH, and R² isCH₃ (acetamidomethyl-cysteine, C(Acm)); R¹ is CH₂, X is NH, and R² isCH₃ (acetyldiaminobutanoic acid, Dab(Ac)); R¹ is C₀ alkyl, X is NH, R²is NHR⁴, and R⁴ is H (carbamoyldiaminopropanoic acid, Dap(urea)); or R¹is CH₂—CH₂, X is NH, and R² is CH₃ (acetylornithine, Orn(Ac)). Inexemplary embodiments of Structure II, R¹ is CH₂, Y is NHR⁴, and R⁴ isCH₃ (methylglutamine, Q(Me)); In exemplary embodiments of Structure III,R¹ is CH₂ and R⁴ is H (methionine-sulfoxide, M(O)); In specificembodiments, the amino acid at position 3 is substituted with Dab(Ac)For example, glucagon agonists can comprise the amino acid sequence ofSEQ ID NO: 595, SEQ ID NO: 601 SEQ ID NO: 603, SEQ ID NO: 604, SEQ IDNO: 605, and SEQ ID NO: 606.

In another embodiment analogs of glucagon are provided that haveenhanced or retained potency at the glucagon receptor relative to thenative glucagon peptide, but also have greatly enhanced activity at theGLP-1 receptor. Glucagon normally has about 1% of the activity ofnative-GLP-1 at the GLP-1 receptor, while GLP-1 normally has less thanabout 0.01% of the activity of native glucagon at the glucagon receptor.Enhanced activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester. In one embodiment, these glucagonanalogs comprise a sequence of SEQ ID NO: 20 wherein the carboxyterminal amino acid has an amide group in place of the carboxylic acidgroup found on the native amino acid. These glucagon analogs have strongactivity at both the glucagon and GLP-1 receptors and thus act asco-agonists at both receptors. In accordance with one embodiment aglucagon and GLP-1 receptor co-agonist is provided wherein the peptidecomprises the sequence of SEQ ID NO: 20, wherein the amino acid atposition 28 is Asn or Lys and the amino acid at position 29 isThr-amide.

Enhanced activity at the GLP-1 receptor is also provided by stabilizingthe alpha-helix structure in the C-terminal portion of glucagon (aroundamino acids 12-29), through formation of an intramolecular bridgebetween the side chains of two amino acids that are separated by threeintervening amino acids, i.e., an amino acid at position “i” and anamino acid at position “i+4”, wherein i is any integer between 12 and25, by two intervening amino acids, i.e., an amino acid at position “j”and an amino acid at position “j+3,” wherein j is any integer between 12and 27, or by six intervening amino acids, i.e., an amino acid atposition “k” and an amino acid at position “k+7,” wherein k is anyinteger between 12 and 22. In exemplary embodiments, the bridge orlinker is about 8 (or about 7-9) atoms in length and forms between sidechains of amino acids at positions 12 and 16, or at positions 16 and 20,or at positions 20 and 24, or at positions 24 and 28. The side chains ofthese amino acids can be linked to one another through non-covalentbonds, e.g., hydrogen-bonding or ionic interactions, such as theformation of salt bridges, or by covalent bonds.

In accordance with one embodiment a glucagon agonist is providedcomprising a glucagon peptide of SEQ ID NO: 20, wherein a lactam ring isformed between the side chains of a lysine residue, located at position12, 20 or 28, and a glutamic acid residue, located at position 16 or 24,wherein the two amino acids of the glucagon peptide whose side chainsparticipate in forming the lactam ring are spaced from one another bythree intervening amino acids. In accordance with one embodiment thelactam bearing glucagon analog comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQID NO: 18. In one embodiment the carboxy terminal amino acid of thelactam bearing peptide comprises an amide group or an ester group inplace of the terminal carboxylic acid. In one embodiment a glucagonpeptide of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18further comprises an additional amino acid covalently bound to thecarboxy terminus of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. Ina further embodiment a glucagon peptide is provided comprising asequence selected from the group consisting of SEQ ID NO: 66, SEQ ID NO:67, SEQ ID NO: 68 and SEQ ID NO: 69 further comprises an additionalamino acid covalently bound to the carboxy terminus of SEQ ID NO: 66,SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69. In one embodiment theamino acid at position 28 is asparagine or lysine and the amino acid atposition 29 is threonine.

In some specific embodiments, stabilization of the alpha helix structurein the C-terminal portion of the glucagon agonist peptide is achievedthrough the formation of a covalent intramolecular bridge other than alactam bridge. For example, suitable covalent bonding methods (i.e.,means of forming a covalent intramolecular bridge) include any one ormore of olefin metathesis, lanthionine-based cyclization, disulfidebridge or modified sulfur-containing bridge formation, the use ofα,ω-diaminoalkane tethers, the formation of metal-atom bridges, andother means of peptide cyclization are used to stabilize the alphahelix.

Enhanced activity at the GLP-1 receptor is also provided by stabilizingthe alpha-helix structure in the C-terminal portion of the glucagonpeptide (around amino acids 12-29) through introduction of one or moreα,α-disubstituted amino acids at positions that retain the desiredactivity. In some aspects, stabilization of the alpha-helix isaccomplished in this manner without purposeful introduction of anintramolecular bridge such as a salt bridge or covalent bond. Suchpeptides may be considered herein as a peptide lacking an intramolecularbridge. In specific aspects, stabilization of the alpha-helix isaccomplished by introducing one or more α,α-disubstituted amino acidswithout introduction of a covalent intramolecular bridge, e.g., a lactambridge, a disulfide bridge. Such peptides may be considered herein as apeptide lacking a covalent intramolecular bridge. In some embodiments,one, two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or29 of a glucagon peptide is substituted with an α,α-disubstituted aminoacid. For example, substitution of position 16 of the glucagon peptidewith amino iso-butyric acid (AIB) enhances GLP-1 activity, in theabsence of a salt bridge or lactam. In some embodiments, one, two, threeor more of positions 16, 20, 21 or 24 are substituted with AIB.

Enhanced activity at the GLP-1 and glucagon receptors for glucagonanalog peptides lacking an intramolecular bridge (e.g., a covalentintramolecular bridge) is provided by the addition of an acyl or alkylgroup to the side chain of the amino acid at position 10 of the peptide.In some aspects, the acyl or alkyl group is not naturally-occurring onan amino acid. In specific aspects, the acyl or alkyl group isnon-native to any naturally-occurring amino acid. In some embodiments,the acyl group is a fatty acyl group, e.g., a C4 to C30 fatty acylgroup. For example, provided herein is a glucagon analog peptide lackinga covalent intramolecular bridge comprising AIB at position 16 and aC14, C16, or C18 fatty acyl group covalently attached to a Lys residueat position 10. Also provided is a glucagon analog peptide lacking anintramolecular bridge (e.g., a covalent intramolecular bridge)comprising AIB at positions 2 and 16 and a C14, C16, or C18 fatty acylgroup covalently attached to a Lys residue at position 10. Such acylatedglucagon analog peptides lacking an intramolecular bridge (e.g., acovalent intramolecular bridge) may be pegylated as further describedherein.

A further enhancement in GLP-1 activity and glucagon activity foracylated glucagon analog peptides lacking an intramolecular bridge(e.g., an intramolecular bridge) may be achieved by incorporating aspacer between the acyl or alkyl group and the side chain of the aminoacid at position 10. In accordance with some embodiments, the spacer(e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional spacer, or a hydrophobic bifunctional spacer) is 3 to 10atoms (e.g., 6 to 10 atoms) in length. In accordance with certainspecific embodiments, the total length of the spacer and acyl or alkylgroup is 14 to 28 atoms, e.g., 17 to 28, 19 to 26 atoms, 19 to 21 atoms.Suitable spacers for purposes of enhancing GLP-1 activity and glucagonactivity for acylated or alkylated peptides lacking an intramolecularbridge (e.g., a covalent intramolecular bridge) are further describedherein.

For example, provided herein is a non-native glucagon peptide thatdiffers from SEQ ID NO: 1 by no more than 10 amino acid modifications,comprising an acyl group or alkyl group, wherein the acyl or alkyl groupis attached to a spacer and the spacer is attached to the side chain ofan amino acid at position 10 of the glucagon peptide, wherein, when saidglucagon peptide lacks a hydrophilic moiety, e.g., PEG, said glucagonpeptide exhibits at least 20% (e.g., at least 30%, at least 40%, atleast 50%, at least 60%, at least 75%, at least 80%, at least 90% atleast 95%, at least 98%, at least 99%, about 100%, about 150%, about200%, about 400%, about 500% or more) of the activity of native GLP-1 atthe GLP-1 receptor. In some embodiments, the glucagon peptide exhibitsat least 0.5% (e.g., at least 1%, at least 2%, at least 3%, at least 4%,at least 5%, at least 10%, at least 20%) of the activity of nativeglucagon at the glucagon receptor, when the glucagon peptide lacks ahydrophilic moiety, e.g., PEG. In some embodiments, the glucagonpeptides described above may exhibit any of the above indicatedactivities and no more than 1000%, 10,000%, 100,000%, or 1,000,000% ofthe activity of native glucagon at the glucagon receptor. In someembodiments, the glucagon peptides described above may exhibit any ofthe above indicated activities and no more than 1000%, 10,000%,100,000%, or 1,000,000% of the activity of native GLP-1 at the GLP-1receptor.

Enhanced activity at the GLP-1 receptor is also provided by an aminoacid modification at position 20. In one embodiment, the glutamine atposition 20 is replaced with another hydrophilic amino acid having aside chain that is either charged or has an ability to hydrogen-bond,and is at least about 5 (or about 4-6) atoms in length, for example,lysine, citrulline, arginine, or ornithine.

GLP-1 activity may be reduced by comprising (i) a C-terminal alphacarboxylate group, (ii) a substitution of the Thr at position 7 with anamino acid lacking a hydroxyl group, e.g., Abu or Ile, (iii) a deletionof the amino acid(s) C-terminal to the amino acid at position 27 or 28(e.g., deletion of the amino acid at position 28, deletion of the aminoacid at positions 28 and 29) to yield a peptide 27 or 28 amino acids inlength, or (iv) a combination thereof.

Any of the modifications described above which increase or decreaseglucagon receptor activity and which increase GLP-1 receptor activitycan be applied individually or in combination. Combinations of themodifications that increase GLP-1 receptor activity may provide higherGLP-1 activity than any of such modifications taken alone. For example,the invention provides glucagon analogs that comprise modifications atposition 16, at position 20, and at the C-terminal carboxylic acidgroup, optionally with a covalent bond between the amino acids atpositions 16 and 20; glucagon analogs that comprise modifications atposition 16 and at the C-terminal carboxylic acid group; glucagonanalogs that comprise modifications at positions 16 and 20, optionallywith a covalent bond between the amino acids at positions 16 and 20; andglucagon analogs that comprise modifications at position 20 and at theC-terminal carboxylic acid group; optionally with the proviso that theamino acid at position 12 is not Arg; or optionally with the provisothat the amino acid at position 9 is not Glu.

Other modifications at position 1 or 2, as described herein, canincrease the peptide's resistance to dipeptidyl peptidase IV (DPP IV)cleavage. For example, the amino acid at position 2 may be substitutedwith D-serine, D-alanine, valine, glycine, N-methyl serine, N-methylalanine, or amino isobutyric acid. Alternatively, or in addition, theamino acid at position 1 may be substituted with D-histidine,desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine,N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, oralpha,alpha-dimethyl imidiazole acetic acid (DMIA).

It was observed that modifications at position 2 (e.g. AIB at position2) and in some cases modifications at position 1 may reduce glucagonactivity, sometimes significantly; surprisingly, this reduction inglucagon activity can be restored by stabilizing the alpha-helix in theC-terminal portion of glucagon, e.g. through a covalent bond betweenamino acids at positions “i” and “i+4”, e.g., 12 and 16, 16 and 20, or20 and 24. In some embodiments, this covalent bond is a lactam bridgebetween a glutamic acid at position 16 and a lysine at position 20. Insome embodiments, this covalent bond is an intramolecular bridge otherthan a lactam bridge. For example, suitable covalent bonding methodsinclude any one or more of olefin metathesis, lanthionine-basedcyclization, disulfide bridge or modified sulfur-containing bridgeformation, the use of α,ω-diaminoalkane tethers, the formation ofmetal-atom bridges, and other means of peptide cyclization.

Glucagon peptides with GLP-1 activity that contain a non-conservativesubstitution of His at position 1 with a large, aromatic amino acid(e.g., Tyr) can retain GLP-1 activity provided that the alpha-helix isstabilized via an intramolecular bridge, e.g. through a covalent bondbetween amino acids at positions “i” and “i+4”, e.g., 12 and 16, 16 and20, or 20 and 24. In some embodiments, this covalent bond is a lactambridge between a glutamic acid at position 16 and a lysine at position20. In some embodiments, this covalent bond is an intramolecular bridgeother than a lactam bridge. For example, suitable covalent bondingmethods include any one or more of olefin metathesis, lanthionine-basedcyclization, disulfide bridge or modified sulfur-containing bridgeformation, the use of α,ω-diaminoalkane tethers, the formation ofmetal-atom bridges, and other means of peptide cyclization.

In yet further exemplary embodiments, any of the foregoing compounds canbe further modified to improve stability by modifying the amino acid atposition 15 of SEQ ID NO: 1 to reduce degradation of the peptide overtime, especially in acidic or alkaline buffers.

In another embodiment the solubility of the glucagon peptides disclosedherein are enhanced by the covalent linkage of a hydrophilic moiety tothe peptide. In one embodiment the hydrophilic moiety is a polyethyleneglycol (PEG) chain, optionally linked to the peptide at one or more ofpositions 16, 17, 21, 24, 29, within a C-terminal extension, or at theC-terminal amino acid. In some embodiments, the native amino acid atthat position is substituted with an amino acid having a side chainsuitable for crosslinking with hydrophilic moieties, to facilitatelinkage of the hydrophilic moiety to the peptide. In other embodiments,an amino acid modified to comprise a hydrophilic group is added to thepeptide at the C-terminal amino acid. In one embodiment the peptideco-agonist comprises a sequence selected from the group consisting ofSEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19wherein the side chain of an amino acid residue at one of position 16,17, 21 or 24 of said glucagon peptide further comprises a polyethyleneglycol chain, having a molecular weight selected from the range of about500 to about 40,000 Daltons. In one embodiment the polyethylene glycolchain has a molecular weight selected from the range of about 500 toabout 5,000 Daltons. In another embodiment the polyethylene glycol chainhas a molecular weight of about 10,000 to about 20,000 Daltons. In yetother exemplary embodiments the polyethylene glycol chain has amolecular weight of about 20,000 to about 40,000 Daltons.

In another embodiment the solubility of any of the preceding glucagonanalogs can be improved by amino acid substitutions and/or additionsthat introduce a charged amino acid into the C-terminal portion of thepeptide, preferably at a position C-terminal to position 27 of SEQ IDNO: 1. Optionally, one, two or three charged amino acids may beintroduced within the C-terminal portion, preferably C-terminal toposition 27. In accordance with one embodiment the native amino acid(s)at positions 28 and/or 29 are substituted with a charged amino acids,and/or in a further embodiment one to three charged amino acids are alsoadded to the C-terminus of the peptide. In exemplary embodiments, one,two or all of the charged amino acids are negatively charged. Additionalmodifications, e.g. conservative substitutions, may be made to theglucagon peptide that still allow it to retain glucagon activity. In oneembodiment an analog of the peptide of SEQ ID NO: 20 is provided whereinthe analog differs from SEQ ID NO: 20 by 1 to 2 amino acid substitutionsat positions 17-26, and in one embodiment the analog differs from thepeptide of SEQ ID NO: 20 by an amino acid substitution at position 20.

In accordance with some embodiments, the glucagon peptides disclosedherein are modified by truncation of the C-terminus by one or two aminoacid residues. Such modified glucagon peptides, as shown herein, retainsimilar activity and potency at the glucagon receptor and GLP-1receptor. In this regard, the glucagon peptides can comprise amino acids1-27 or 1-28 of the native glucagon peptide (SEQ ID NO: 1), optionallywith any of the additional modifications described herein.

In accordance with one embodiment the glucagon peptides disclosed hereinare modified by the addition of a second peptide to the carboxy terminusof the glucagon peptide, for example, SEQ ID NO: 26, SEQ ID NO: 27 orSEQ ID NO: 28. In one embodiment a glucagon peptide having a peptidesequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, and SEQ ID NO: 69 is covalently bound through a peptide bondto a second peptide, wherein the second peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28. In a further embodiment, in glucagon peptides whichcomprise the C-terminal extension, the threonine at position 29 of thenative glucagon peptide is replaced with a glycine. A glucagon analoghaving a glycine substitution for threonine at position 29 andcomprising the carboxy terminal extension of SEQ ID NO: 26 is four timesas potent at the GLP-1 receptor as native glucagon modified to comprisethe carboxy terminal extension of SEQ ID NO: 26. Potency at the GLP-1receptor can be further enhanced by an alanine substitution for thenative arginine at position 18.

Any of the glucagon peptides disclosed herein can be modified tocomprise an acyl group or alkyl group, e.g., a C4 to C30 acyl or alkylgroup. In some aspects, the acyl group or alkyl group is non-native toany naturally-occurring amino acid.

Acylation or alkylation can increase the half-life of the glucagonpeptides in circulation. Acylation or alkylation can advantageouslydelay the onset of action and/or extend the duration of action at theglucagon and/or GLP-1 receptors and/or improve resistance to proteasessuch as DPP-IV. As shown herein, the activity at the glucagon receptorand GLP-1 receptor of the glucagon peptide is maintained, if notsubstantially enhanced, after acylation. Further, the potency of theacylated glucagon peptides were comparable to the unacylated versions ofthe glucagon peptides, if not substantially enhanced. Glucagon peptidesmay be acylated or alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position. Insome embodiments, the invention provides a glucagon peptide modified tocomprise an acyl group or alkyl group covalently linked to the aminoacid at position 10 of the glucagon peptide. The glucagon peptide mayfurther comprise a spacer between the amino acid at position 10 of theglucagon peptide and the acyl group or alkyl group. In some embodiments,the acyl group is a fatty acid or bile acid, or salt thereof, e.g. a C4to C30 fatty acid, a C8 to C24 fatty acid, cholic acid, a C4 to C30alkyl, a C8 to C24 alkyl, or an alkyl comprising a steroid moiety of abile acid. The spacer is any moiety with suitable reactive groups forattaching acyl or alkyl groups. In exemplary embodiments, the spacercomprises an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional spacer, or a hydrophobic bifunctional spacer. In someembodiments, the spacer is selected from the group consisting of: Trp,Glu, Asp, Cys and a spacer comprising NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH,wherein m is any integer from 1 to 6 and n is any integer from 2 to 12.Such acylated or alkylated glucagon peptides may also further comprise ahydrophilic moiety, optionally a polyethylene glycol. Any of theforegoing glucagon peptides may comprise two acyl groups or two alkylgroups, or a combination thereof.

Thus, as disclosed herein high potency glucagon analogs or glucagonco-agonist analogs are provided that also exhibit improved solubilityand/or stability. An exemplary high potency glucagon analog exhibits atleast about 200% of the activity of native glucagon at the glucagonreceptor, and optionally is soluble at a concentration of at least 1mg/mL at a pH between 6 and 8, or between 6 and 9, or between 7 and 9(e.g. pH 7), and optionally retains at least 95% of the original peptide(e.g. 5% or less of the original peptide is degraded or cleaved) after24 hours at 25° C. As another example, an exemplary glucagon co-agonistanalog exhibits greater than about 40% or greater than about 60%activity at both the glucagon and the GLP-1 receptors (at a ratiobetween about 1:3 and 3:1, or between about 1:2 and 2:1), is optionallysoluble at a concentration of at least 1 mg/mL at a pH between 6 and 8or between 6 and 9, or between 7 and 9 (e.g. pH 7), and optionallyretains at least 95% of the original peptide after 24 hours at 25° C.Another exemplary glucagon co-agonist analog exhibits about 175% or moreof the activity of native glucagon at the glucagon receptor and about20% or less of the activity of native GLP-1 at the GLP-1 receptor, isoptionally soluble at a concentration of at least 1 mg/mL at a pHbetween 6 and 8 or between 6 and 9, or between 7 and 9 (e.g. pH 7), andoptionally retains at least 95% of the original peptide after 24 hoursat 25° C. Yet another exemplary glucagon co-agonist analog exhibitsabout 10% or less of the activity of native glucagon at the glucagonreceptor and at least about 20% of the activity of native GLP-1 at theGLP-1 receptor, is optionally soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7 and 9(e.g. pH 7), and optionally retains at least 95% of the original peptideafter 24 hours at 25° C. Yet another exemplary glucagon co-agonistanalog exhibits about 10% or less but above 0.1%, 0.5% or 1% of theactivity of native glucagon at the glucagon receptor and at least about50%, 60%, 70%, 80%, 90% or 100% or more of the activity of native GLP-1at the GLP-1 receptor, is optionally soluble at a concentration of atleast 1 mg/mL at a pH between 6 and 8 or between 6 and 9, or between 7and 9 (e.g. pH 7), and optionally retains at least 95% of the originalpeptide after 24 hours at 25° C. In some embodiments, the glucagonpeptides exhibit no more than about 100%, 1000%, 10,000%, 100,000%, or1,000,000% of the activity of native GLP-1 at the GLP-1 receptor. Insome embodiments, such glucagon analogs retain at least 22, 23, 24, 25,26, 27 or 28 of the naturally occurring amino acids at the correspondingpositions in native glucagon (e.g. have 1-7, 1-5 or 1-3 modificationsrelative to naturally occurring glucagon).

Any one of the following peptides is excluded from the compounds of theinvention, although any of the following peptides comprising one or morefurther modifications thereto as described herein exhibiting the desiredGLP-1 or co-agonist activity, pharmaceutical compositions, kits, andtreatment methods using such compounds may be included in the invention:The peptide of SEQ ID NO: 1 with an [Arg12] substitution and with aC-terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys20]substitutions and with a C-terminal amide; The peptide of SEQ ID NO: 1with [Arg12,Lys24] substitutions and with a C-terminal amide; Thepeptide of SEQ ID NO: 1 with [Arg12,Lys29] substitutions and with aC-terminal amide; The peptide of SEQ ID NO: 1 with a [Glu9]substitution; The peptide of SEQ ID NO: 1 missing His1, with [Glu9,Glu16, Lys29] substitutions and C-terminal amide; The peptide of SEQ IDNO: 1 with [Glu9, Glu16, Lys29] substitutions and with a C-terminalamide; The peptide of SEQ ID NO: 1 with [Lys13, Glu17] substitutionslinked via lactam bridge and with a C-terminal amide; The peptide of SEQID NO: 1 with [Lys17, Glu21] substitutions linked via lactam bridge andwith a C-terminal amide; The peptide of SEQ ID NO: 1 missing His1, with[Glu20, Lys24] substitutions linked via lactam bridge.

In accordance with one embodiment a pharmaceutical composition isprovided comprising any of the novel glucagon peptides disclosed herein,preferably sterile and preferably at a purity level of at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. Such compositions may containa glucagon peptide at a concentration of at least A, wherein A is 0.001mg/ml, 0.01 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml,12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml orhigher. In other embodiments, such compositions may contain a glucagonpeptide at a concentration of at most B, wherein B is 30 mg/ml, 25mg/ml, 24 mg/ml, 23, mg/ml, 22 mg/ml, 21 mg/ml, 20 mg/ml, 19 mg/ml, 18mg/ml, 17 mg/ml, 16 mg/ml, 15 mg/ml, 14 mg/ml, 13 mg/ml, 12 mg/ml, 11mg/ml 10 mg/ml, 9 mg/ml, 8 mg/ml, 7 mg/ml, 6 mg/ml, 5 mg/ml, 4 mg/ml, 3mg/ml, 2 mg/ml, 1 mg/ml, or 0.1 mg/ml. In some embodiments, thecompositions may contain a glucagon peptide at a concentration range ofA to B mg/ml, for example, 0.001 to 30.0 mg/ml. In one embodiment thepharmaceutical compositions comprise aqueous solutions that aresterilized and optionally stored within various containers. Thecompounds of the present invention can be used in accordance with oneembodiment to prepare pre-formulated solutions ready for injection. Inother embodiments the pharmaceutical compositions comprise a lyophilizedpowder. The pharmaceutical compositions can be further packaged as partof a kit that includes a disposable device for administering thecomposition to a patient. The containers or kits may be labeled forstorage at ambient room temperature or at refrigerated temperature.

In accordance with one embodiment a method of rapidly increasing glucoselevel or treating hypoglycemia using a pre-formulated aqueouscomposition of glucagon peptides of the invention is provided. Themethod comprises the step of administering an effective amount of anaqueous solution comprising a novel modified glucagon peptide of thepresent disclosure. In one embodiment the glucagon peptide is pegylatedat position 21 or 24 of the glucagon peptide and the PEG chain has amolecular weight of about 500 to about 5,000 Daltons. In one embodimentthe modified glucagon solution is prepackaged in a device that is usedto administer the composition to the patient suffering fromhypoglycemia.

In accordance with one embodiment an improved method of regulating bloodglucose levels in insulin dependent patients is provided. The methodcomprises the steps of administering insulin in an amounttherapeutically effective for the control of diabetes and administeringa novel modified glucagon peptide of the present disclosure in an amounttherapeutically effective for the prevention of hypoglycemia, whereinsaid administering steps are conducted within twelve hours of eachother. In one embodiment the glucagon peptide and the insulin areco-administered as a single composition, wherein the glucagon peptide ispegylated with a PEG chain having a molecular weight selected from therange of about 5,000 to about 40,000 Daltons

In another embodiment a method is provided for inducing the temporaryparalysis of the intestinal tract. The method comprises the step ofadministering one or more of the glucagon peptides disclosed herein to apatient.

Metabolic Syndrome, also known as metabolic syndrome X, insulinresistance syndrome or Reaven's syndrome, is a disorder that affectsover 50 million Americans. Metabolic Syndrome is typically characterizedby a clustering of at least three or more of the following risk factors:(1) abdominal obesity (excessive fat tissue in and around the abdomen),(2) atherogenic dyslipidemia (blood fat disorders including hightriglycerides, low HDL cholesterol and high LDL cholesterol that enhancethe accumulation of plaque in the artery walls), (3) elevated bloodpressure, (4) insulin resistance or glucose intolerance, (5)prothrombotic state (e.g. high fibrinogen or plasminogen activatorinhibitor-1 in blood), and (6) pro-inflammatory state (e.g. elevatedC-reactive protein in blood). Other risk factors may include aging,hormonal imbalance and genetic predisposition.

Metabolic Syndrome is associated with an increased the risk of coronaryheart disease and other disorders related to the accumulation ofvascular plaque, such as stroke and peripheral vascular disease,referred to as atherosclerotic cardiovascular disease (ASCVD). Patientswith Metabolic Syndrome may progress from an insulin resistant state inits early stages to full blown type II diabetes with further increasingrisk of ASCVD. Without intending to be bound by any particular theory,the relationship between insulin resistance, Metabolic Syndrome andvascular disease may involve one or more concurrent pathogenicmechanisms including impaired insulin-stimulated vasodilation, insulinresistance-associated reduction in NO availability due to enhancedoxidative stress, and abnormalities in adipocyte-derived hormones suchas adiponectin (Lteif and Mather, Can. J. Cardiol. 20 (suppl. B):66B-76B(2004)).

According to the 2001 National Cholesterol Education Program AdultTreatment Panel (ATP III), any three of the following traits in the sameindividual meet the criteria for Metabolic Syndrome: (a) abdominalobesity (a waist circumference over 102 cm in men and over 88 cm inwomen); (b) serum triglycerides (150 mg/dl or above); (c) HDLcholesterol (40 mg/dl or lower in men and 50 mg/dl or lower in women);(d) blood pressure (130/85 or more); and (e) fasting blood glucose (110mg/dl or above). According to the World Health Organization (WHO), anindividual having high insulin levels (an elevated fasting blood glucoseor an elevated post meal glucose alone) with at least two of thefollowing criteria meets the criteria for Metabolic Syndrome: (a)abdominal obesity (waist to hip ratio of greater than 0.9, a body massindex of at least 30 kg/m², or a waist measurement over 37 inches); (b)cholesterol panel showing a triglyceride level of at least 150 mg/dl oran HDL cholesterol lower than 35 mg/dl; (c) blood pressure of 140/90 ormore, or on treatment for high blood pressure). (Mather, Ruchi,“Metabolic Syndrome,” ed. Shiel, Jr., William C., MedicineNet.com, May11, 2009).

For purposes herein, if an individual meets the criteria of either orboth of the criteria set forth by the 2001 National CholesterolEducation Program Adult Treatment Panel or the WHO, that individual isconsidered as afflicted with Metabolic Syndrome.

Without being bound to any particular theory, glucagon peptidesdescribed herein are useful for treating Metabolic Syndrome.Accordingly, the invention provides a method of preventing or treatingMetabolic Syndrome, or reducing one, two, three or more risk factorsthereof, in a subject, comprising administering to the subject aglucagon peptide described herein in an amount effective to prevent ortreat Metabolic Syndrome, or the risk factor thereof.

Nonalcoholic fatty liver disease (NAFLD) refers to a wide spectrum ofliver disease ranging from simple fatty liver (steatosis), tononalcoholic steatohepatitis (NASH), to cirrhosis (irreversible,advanced scarring of the liver). All of the stages of NAFLD have incommon the accumulation of fat (fatty infiltration) in the liver cells(hepatocytes). Simple fatty liver is the abnormal accumulation of acertain type of fat, triglyceride, in the liver cells with noinflammation or scarring. In NASH, the fat accumulation is associatedwith varying degrees of inflammation (hepatitis) and scarring (fibrosis)of the liver. The inflammatory cells can destroy the liver cells(hepatocellular necrosis). In the terms “steatohepatitis” and“steatonecrosis”, steato refers to fatty infiltration, hepatitis refersto inflammation in the liver, and necrosis refers to destroyed livercells. NASH can ultimately lead to scarring of the liver (fibrosis) andthen irreversible, advanced scarring (cirrhosis). Cirrhosis that iscaused by NASH is the last and most severe stage in the NAFLD spectrum.(Mendler, Michel, “Fatty Liver: Nonalcoholic Fatty Liver Disease (NAFLD)and Nonalcoholic Steatohepatitis (NASH),” ed. Schoenfield, Leslie J.,MedicineNet.com, Aug. 29, 2005).

Alcoholic Liver Disease, or Alcohol-Induced Liver Disease, encompassesthree pathologically distinct liver diseases related to or caused by theexcessive consumption of alcohol: fatty liver (steatosis), chronic oracute hepatitis, and cirrhosis. Alcoholic hepatitis can range from amild hepatitis, with abnormal laboratory tests being the only indicationof disease, to severe liver dysfunction with complications such asjaundice (yellow skin caused by bilirubin retention), hepaticencephalopathy (neurological dysfunction caused by liver failure),ascites (fluid accumulation in the abdomen), bleeding esophageal varices(varicose veins in the esophagus), abnormal blood clotting and coma.Histologically, alcoholic hepatitis has a characteristic appearance withballooning degeneration of hepatocytes, inflammation with neutrophilsand sometimes Mallory bodies (abnormal aggregations of cellularintermediate filament proteins). Cirrhosis is characterized anatomicallyby widespread nodules in the liver combined with fibrosis. (Worman,Howard J., “Alcoholic Liver Disease”, Columbia University Medical Centerwebsite).

Without being bound to any particular theory, glucagon peptidesdescribed herein are useful for the treatment of Alcoholic LiverDisease, NAFLD, or any stage thereof, including, for example, steatosis,steatohepatitis, hepatitis, hepatic inflammation, NASH, cirrhosis, orcomplications thereof. Accordingly, the invention provides a method ofpreventing or treating Alcoholic Liver Disease, NAFLD, or any stagethereof, in a subject comprising administering to a subject a glucagonpeptide described herein in an amount effective to prevent or treatAlcoholic Liver Disease, NAFLD, or the stage thereof. Such treatmentmethods include reduction in one, two, three or more of the following:liver fat content, incidence or progression of cirrhosis, incidence ofhepatocellular carcinoma, signs of inflammation, e.g. abnormal hepaticenzyme levels (e.g., aspartate aminotransferase AST and/or alanineaminotransferase ALT, or LDH), elevated serum ferritin, elevated serumbilirubin, and/or signs of fibrosis, e.g. elevated TGF-beta levels. Inpreferred embodiments, the glucagon peptides are used treat patients whohave progressed beyond simple fatty liver (steatosis) and exhibit signsof inflammation or hepatitis. Such methods may result, for example, inreduction of AST and/or ALT levels.

In yet another embodiment a method of treating hyperglycemia, or amethod of reducing weight gain or inducing weight loss is provided,which involves administering an effective amount of an aqueous solutioncomprising a glucagon peptide of the invention. In one embodiment eithermethod comprises administering an effective amount of a compositioncomprising a glucagon agonist selected from the group consisting of SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. Inanother embodiment, the method comprises administering an effectiveamount of a composition comprising a glucagon agonist, wherein theglucagon agonist comprising a glucagon peptide selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69,wherein amino acid 29 of the glucagon peptide is bound to a secondpeptide through a peptide bond, and said second peptide comprises thesequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28. In furtherembodiments, methods of treating diabetes involving co-administering aconventional dose or a reduced dose of insulin and a glucagon peptide ofthe invention are provided. Methods of treating diabetes with a glucagonpeptide of the invention, without co-administering insulin are alsoprovided.

In yet another aspect, the invention provides novel methods for treatinghyperglycemia and novel methods for decreasing appetite or promotingbody weight loss that involve administration of a glucagon/GLP-1co-agonist molecule (including pharmaceutically acceptable saltsthereof) that activates both the glucagon receptor and the GLP-1receptor. Agonism, i.e., activation, of both the glucagon and GLP-1receptors provides an unexpected improvement compared to GLP-1 agonismalone in treating hyperglycemia. Thus, the addition of glucagon agonismprovides an unexpected additive or synergistic effect, or otherunexpected clinical benefit(s). Administration with a conventional doseof insulin, a reduced dose of insulin, or without insulin iscontemplated according to such methods. Agonism of the glucagon receptoralso has an unexpected beneficial effect compared to GLP-1 agonism alonein promoting weight loss or preventing weight gain.

Exemplary glucagon/GLP-1 co-agonist molecules include glucagon peptidesof the invention, GLP-1 analogs that activate both GLP-1 and glucagonreceptors, fusions of glucagon and GLP-1, or fusions of glucagon analogsand GLP-1 analogs, or chemically modified derivatives thereof.Alternatively, a compound that activates the glucagon receptor can beco-administered with a compound that activates the GLP-1 receptor (suchas a GLP-1 analog, an exendin-4 analog, or derivatives thereof). Theinvention also contemplates co-administration of a glucagon agonistanalog with a GLP-1 agonist analog.

Such methods for treating hyperglycemia and/or for decreasing appetiteor promoting body weight loss include administration of a glucagonanalog with a modification at position 12 (e.g. Arg12), optionally incombination with modifications at position 16 and/or 20. The methods ofthe invention also include administration of glucagon analogs comprisingan intramolecular bridge between the side chains of two amino acidswithin the region of amino acids 12 and 29 that are separated by threeintervening amino acids, e.g. positions 12 and 16, positions 13 and 17(e.g., Lys13 Glu17 or Glu13 Lys17), positions 16 and 20, positions 17and 21 (e.g. Lys17 Glu 21 or Glu17 Lys 21), positions 20 and 24, orpositions 24 and 28, with the optional proviso that the amino acid atposition 9 is not Glu, and optionally including a C-terminal amide orester.

In accordance with one embodiment excluded from such glucagon/GLP-1co-agonist molecules are any glucagon analogs or GLP-1 analogs in theprior art known to be useful in such a method. In another embodimentpeptides described in U.S. Pat. No. 6,864,069 as acting as both a GLP-1agonist and a glucagon antagonist for treating diabetes are alsoexcluded as glucagon/GLP-1 co-agonist molecules. In another embodiment,excluded is the use of glucagon antagonists to treat diabetes, such asthe antagonists described in Unson et al., J. Biol. Chem., 264:789-794(1989), Ahn et al., J. Med. Chem., 44:3109-3116 (2001), and Sapse etal., Mol. Med., 8(5):251-262 (2002). In a further embodimentoxyntomodulin or a glucagon analog that contains the 8 C-terminal aminoacids of oxyntomodulin (SEQ ID NO: 27) are also excluded asglucagon/GLP-1 co-agonist molecules.

Such methods for treating hyperglycemia are expected to be useful for avariety of types of hyperglycemia, including diabetes, diabetes mellitustype I, diabetes mellitus type II, or gestational diabetes, eitherinsulin-dependent or non-insulin-dependent, and reducing complicationsof diabetes including nephropathy, retinopathy and vascular disease.Such methods for reducing appetite or promoting loss of body weight areexpected to be useful in reducing body weight, preventing weight gain,or treating obesity of various causes, including drug-induced obesity,and reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases.

All therapeutic methods, pharmaceutical compositions, kits and othersimilar embodiments described herein contemplate that the use of theterm glucagon analogs includes all pharmaceutically acceptable salts oresters thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph representing the stability of GlucagonCys²¹maleimidoPEG_(5K) at 37° C. incubated for 24, 48, 72, 96, 144 and166 hours, respectively.

FIG. 2 represents data generated from HPLC analysis of GlucagonCys²¹maleimidoPEG_(5K) at pH 5 incubated at 37° C. for 24, 72 or 144hours, respectively.

FIG. 3 represents data showing receptor mediated cAMP induction byglucagon analogs. More particularly, FIG. 3A compares induction of theglucagon receptor by glucagon analogs E16, K20 , E15, E16 ▴, E16, K20▾, E15, E16

, E16

and Gluc-NH₂ ▪

FIGS. 4A and 4B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 4A compares induction ofthe glucagon receptor by glucagon analogs Gluc-NH₂ , E16Gluc-NH₂ ▴, E3,E16 Gluc-NH₂ ▾, Orn3, E16 Gluc-NH₂

and Nle3, E16 Gluc-NH₂,

relative to native glucagon ▪, whereas FIG. 4B compares induction of theGLP-1 receptor by glucagon analogs Gluc-NH₂ , E16 Gluc-NH₂ ▴, E3,E16Gluc-NH₂ ▾, Orn3, E16 Gluc-NH₂

and Nle3, E16 Gluc-NH₂,

relative to native GLP-1 ▪.

FIGS. 5A and 5B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 5A compares induction ofthe glucagon receptor by glucagon analogs (E16, K20 Gluc-NH₂  (5 nM,stock solution), E15, E16 Gluc-NH₂ ▴ (5 nM, stock solution), E16, K20Gluc-NH₂ ▾ (10 nM, stock solution), E15, E16 Gluc-NH₂

(10 nM, stock solution) and E16 Gluc-NH₂

) relative to glucagon-NH₂ (▪), whereas FIG. 5B compares induction ofthe GLP-1 receptor by glucagon analogs (E16, K20 Gluc-NH₂ , E15, E16Gluc-NH₂ ▴, and E16 Gluc-NH₂,

) relative to GLP-1 (▪) and glucagon-NH₂ (□).

FIGS. 6A and 6B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 6A compares induction ofthe glucagon receptor by glucagon analogs (Gluc-NH₂ , K12E16-NH₂ lactam▴, E16K20-NH₂ lactam ▾, K20E24-NH₂ lactam

and E24K28-NH₂ lactam

) relative to glucagon (▪), whereas FIG. 6B compares induction of theGLP-1 receptor by glucagon analogs (Gluc-NH₂ , K12E16-NH₂ lactam ▴,E16K20-NH₂ lactam ▾, K20E24-NH₂ lactam

and E24K28-NH₂ lactam

) relative to GLP-1 ().

FIGS. 7A and 7B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 7A compares induction ofthe glucagon receptor by glucagon analogs (Gluc-NH₂ , E16 Gluc-NH₂, ▴,K12, E16 Gluc-NH₂ lactam ▾, E16, K20 Gluc-NH₂

and E16, K20 Gluc-NH₂ lactam

) relative to glucagon (▪), whereas FIG. 7B compares induction of theGLP-1 receptor by glucagon analogs (Glue-NH₂ , E16 Gluc-NH₂, ▴, K12,E16 Gluc-NH₂ lactam ▾, E16, K20 Gluc-NH₂

and E16, K20 Gluc-NH₂ lactam

) relative to GLP-1 (▪).

FIGS. 8A-8F represent data showing receptor mediated cAMP induction byglucagon analogs at the glucagon receptor (FIGS. 8A, 8C and 8E) or theGLP-1 receptor (FIGS. 8B, 8C and 8F) wherein hE=homoglutamic acid andhC=homocysteic acid.

FIGS. 9A and 9B: represent data showing receptor mediated cAMP inductionby GLP (17-26) glucagon analogs, wherein amino acid positions 17-26 ofnative glucagon (SEQ ID NO: 1) have been substituted with the aminoacids of positions 17-26 of native GLP-1 (SEQ ID NO: 50). Moreparticularly, FIG. 9A compares induction of the glucagon receptor by thedesignated GLP (17-26) glucagon analogs, and FIG. 9B compares inductionof the GLP-1 receptor by the designated GLP (17-26) glucagon analogs.

FIGS. 10A-E: are graphs providing in vivo data demonstrating the abilityof the glucagon peptides of the present invention to induce weight lossin mice injected subcutaneously with the indicated amounts of therespective compounds. Sequence Identifiers for the glucagon peptidelisted in FIGS. 10A-10E are as follows, for FIG. 10A: Chimera 2 Aib2 C2440K PEG (SEQ ID NO: 486), Aib2 C24 Chimera 2 40K lactam (SEQ ID NO: 504)and Aib2 E16 K20 Gluc-NH2 Lac 40K (SEQ ID NO: 528); FIG. 10B: Aib2 C24Chi 2 lactam 40K (SEQ ID NO: 504), DMIA1 C24 Chi 2 Lactam 40K (SEQ IDNO: 505), Chimera 2 DMIA1 C24 40K (SEQ ID NO: 519), and Chimera 2 Aib2C24 40K (SEQ ID NO: 486), wherein the number at the end of the sequencedesignates the dosage used, either 70 or 350 nmol/kg; FIG. 10C: AIB2w/lactam C24 40K (SEQ ID NO: 504), AIB2 E16 K20 w/lactam C24 40K (SEQ IDNO: 528), DMIA1 E16 K20 w/lactam C24 40K (SEQ ID NO: 510), DMIA1 E16 K20w/lactam CEX 40K (SEQ ID NO: 513) and DMIA1 E16 K20 w/o lactam CEX 40K(SEQ ID NO: 529); FIG. 10D: AIB2 w lactam C24 40K (SEQ ID NO: 504), AIB2E16 K20 w lactam C24 40K (SEQ ID NO: 528), DMIA1 E16 K20 w lactam C2440K (SEQ ID NO: 510) and DMIA1 E16 K20 w lactam/Cex C24 40K (SEQ ID NO:513), wherein the number at the end of the sequence designates thedosage used, either 14 or 70 nmol/kg/wk; FIG. 10E: AIB2 w/o lactam C2440K (SEQ ID NO: 486), Chi 2 AIB2 C24 CEX 40K (SEQ ID NO: 533), AIB2 E16A18 K20 C24 40K (SEQ ID NO: 492), AIB2 w/o lactam CEX G29 C40 40K (SEQID NO: 488), AIB2 w/o lactam CEX C40 C41-2 (SEQ ID NO: 532), AIB2 w/olactam CEX C24 C40-2 (SEQ ID NO: 531) and AIB2 w/o lactam C24 60K (SEQID NO: 498), wherein the designation 40K or 60K represents the molecularweight of the polyethylene chain attached to the glucagon peptide.

FIGS. 11-13 are graphs providing in vivo data demonstrating the abilityof acylated glucagon peptides to induce weight loss (FIG. 11), reducefood intake (FIG. 12), and reduce blood glucose levels (FIG. 13) in miceinjected subcutaneously with the indicated amounts of the compounds.

FIGS. 14A and 14B represent data showing glucagon and GLP-1 receptormediated cAMP induction, respectively, by glucagon analogs.

FIG. 15 represents a graph of blood glucose (mg/dL) as a function oftime (mins) in DIO mice treated with 2 nmol/kg of vehicle only(triangles), Chimera-2 AIB², K¹⁰—C8 Cys²⁴-40 kD PEG (open squares), orChimera-2 AIB², K¹⁰—C16 Cys²⁴-40 kD PEG (inverted triangles) followed byglucose challenge 15 mins after administration of the peptide.

FIG. 16 represents a graph of blood glucose (mg/dL) as a function oftime (mins) in DIO mice treated with 20 nmol/kg of vehicle only(triangles), Chimera-2 AIB², K¹⁰—C8 Cys²⁴-40 kD PEG (open squares), orChimera-2 AIB², K¹⁰—C16 Cys²⁴-40 kD PEG (inverted triangles) followed byglucose challenge 15 mins after administration of the peptide.

FIG. 17 represents a graph of blood glucose (mg/dL) as a function oftime (mins) of DIO mice treated with 70 nmol/kg of vehicle only(inverted triangles), Chimera-2 AIB², K¹⁰—C8 Cys²⁴-40 kD PEG (opentriangles), Chimera-2 AIB², K¹⁰—C16 Cys²⁴-40 kD PEG (diamonds), orChimera-2 AIB², Cys²⁴-40 kD PEG (open squares) followed by glucosechallenge 15 mins after administration of the peptide.

FIG. 18 represents a graph of blood glucose (mg/dL) as a function oftime (mins) of DIO mice treated with 70 nmol/kg of vehicle only(inverted triangles), Chimera-2 AIB², K¹⁰—C8 Cys²⁴-40 kD PEG (opentriangles), Chimera-2 AIB², K¹⁰—C16 Cys²⁴-40 kD PEG (diamonds), orChimera-2 AIB², Cys²⁴-40 kD PEG (open squares) followed by glucosechallenge 24 hours after administration of the peptide.

FIG. 19 represents a graph of the change in body weight (%) as afunction of time (days) in DIO mice treated with 15 or 70 nmol/kg ofvehicle only (diamonds with solid line); Chimera-2 AIB², Cys²⁴-40 kD PEG(15 nmol/kg, open diamonds with dotted line; 70 nmol/kg, open triangleswith solid line); Chimera-2 AIB², K¹⁰⁻C8 Cys²⁴-40 kD PEG (15 nmol/kg,closed triangle with dotted line; 70 nmol/kg, closed triangle with solidline); Chimera-2 AIB², K¹⁰—C16 Cys²⁴-40 kD Peg (15 nmol/kg, invertedtriangle with dotted line; 70 nmol/kg; inverted triangle with solidline).

FIG. 20 represents a graph of the total change in body weight (%) inmice 14 days after QW injections of 10, 20, 40, or 80 nmol/kg Peptide AK¹⁰—C₁₄ or 20 nmol/kg Chimera-2 AIB²¹K¹⁰—C8 Cys²⁴-40 kD or a vehiclecontrol

FIG. 21 represents a graph of the blood glucose levels (mg/dL) inresponse to a glucose injection of mice injected with 10, 20, 40, or 80nmol/kg Peptide A K¹⁰—C₁₄ or 20 nmol/kg Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40kD or a vehicle control 24 hours prior to the glucose injection.

FIG. 22 represents a graph of the total change in body weight (%) ofmice injected with vehicle control, Liraglutide, (C16) Glucagon Amide,γE-γE-C16 Glucagon Amide, AA-C16 Glucagon Amide, or βAβA-C16 GlucagonAmide at the indicated dose.

FIG. 23 represents a graph of the fat mass (g) as measured on Day 7 ofthe study of mice injected with vehicle control, Liraglutide, (C16)Glucagon Amide, γE-γE-C16 Glucagon Amide, AA-C16 Glucagon Amide, orβAβA-C16 Glucagon Amide at the indicated dose.

FIG. 24 represents a graph of the change in blood glucose (mg/dL; Day 7levels minus Day 0 levels) of mice injected with vehicle control,Liraglutide, (C16) Glucagon Amide, γE-γE-C16 Glucagon Amide, AA-C16Glucagon Amide, or PAPA-C16 Glucagon Amide at the indicated dose.

FIG. 25 represents a graph of the mean residue ellipticity as a functionof wavelength (nm) for Peptide X-PEG or Peptide Y-PEG in 10 mM Phosphate(pH 5.9) either with or without 10% TFE.

FIG. 26 represents a graph of the % cAMP produced in response toGlucagon, GLP-1, Peptide X, Peptide X-PEG, Peptide Y, or Peptide Y-PEGbinding to either the glucagon receptor (left) or GLP-1 receptor (right)as a function of peptide concentration (nM).

FIG. 27 represents a collection of graphs which demonstrate the in vivoeffects on A) body weight, B) fat mass, C) food intake, and D) fastingblood glucose levels in diet induced obese mice treated for one weekwith vehicle control, Peptide X-PEG, or Peptide Y-PEG. Morespecifically, FIG. 27A represents a graph of the % change in body weight(BW) as a function of time (days), FIG. 27 B represents a graph of the %change in fat mass as measured on Day 7 (as compared to initial fat massmeasurements), FIG. 27C represents a graph of the total food intake (g)over the course of the study as measured on Day 7, and FIG. 27Drepresents a graph of the change in blood glucose (mg/dL) as measured onDay 7 (in comparison to initial blood glucose levels).

FIG. 28 represents a collection of graphs which demonstrate the in vivoeffects on body weight (FIGS. 28A and 28C) and fasting blood glucoselevels (FIGS. 28B and 28D) in mice treated with either Peptide X-PEG(FIGS. 28A and 28B) or Peptide Y-PEG (FIGS. 28C and 28D) at varyingdoses (nmol/kg/week).

FIG. 29 represents a collection of graphs showing the in vivo effects onA) body weight (BW), B) body fat mass, C) overall food intake, D) energyexpenditure, E) respiratory quotient, F) locomotor activity, G) fastingblood glucose, H) glucose tolerance, and I) total plasma insulin levelsin diet induced obese mice treated for one month with a vehicle control,Peptide X-PEG, or Peptide Y-PEG.

FIG. 30 represents a collection of graphs showing the in vivo Week 3effects on calorimetric measurements of A) food intake, B) total energyexpenditure, C) total respiratory quotient, D) locomotor activity, E)total locomotor activity, F) area under the curve ipGTT, G) plasmaC-peptide levels, H) PEPCK/HPRT fold expression, and I) G6P/HPRT foldexpression levels in diet induced obese mice treated for one month witha vehicle control, Peptide X-PEG, or Peptide Y-PEG

FIG. 31 represents a collection of graphs demonstrating the in vivoeffects on plasma A) cholesterol, B) cholesterol FPLC, C) triglycerides,D) leptin., E) resistin, and F) adiponectin in diet induced obese micetreated for one month with a vehicle control, Peptide X-PEG, or PeptideY-PEG.

FIG. 32 represents a collection of graphs demonstrating the in vivoeffects on A) BAT UCP-1 expression levels and B) white adipose tissue asreflected by phosphorylation of hormone sensitive lipase (pHSL) in micetreated with a vehicle control, Peptide X-PEG, or Peptide Y-PEG

FIG. 33 represents a collection of graphs demonstrating the in vivoeffects of a vehicle control, Peptide X-PEG, or Peptide Y-PEG in DIOrats on A) body weight and B) fat mass. FIG. 33C represents a graph ofthe relative expression of CD68 to TFIIB as quantitatively assessed byreal-time RT-PCR in epidiymal adipose tissue isolated from mice treatedfor two weeks with Peptide Y-PEG, Peptide X-PEG, or vehicle. Data arepresented as relative CD68 mRNA expression normalized to TF1IB mRNAexpression and expressed as mean±SEM.

FIGS. 34A to 34F represent a collection of graphs demonstrating the invivo effects on body weight (BW; 34A and 34B), fat mass (34C), foodintake (34D), and blood glucose levels (34E and 34F) in GLP-1-R knockout mice treated with a vehicle control, Peptide X-PEG, or PeptideY-PEG.

FIGS. 35A to 35C represent a series of graphs demonstrating the in vivoeffects on body weight (35A), blood glucose (35B), and fat mass (35C) inDIO mice treated with vehicle control, Peptide V, or Peptide U.

FIG. 36 represents a graph of the % total change in body weight of miceinjected with vehicle, liraglutide, glucagon amide, MT-261, MT-345,MT-347, or MT-348 at the dose indicated in ( ) as measured on Day 14 ofthe study.

FIG. 37 represents a graph of the % total change in body weight of miceinjected with vehicle, MT-278, MT-358, MT-261, MT-297, or MT-364 asmeasured on Day 7 of the study.

FIG. 38 represents a graph of the % total change in body weight of miceinjected with vehicle only, Peptide 83, Peptide 900, Peptide 901, orMT-364, as measured on Day 7 of the study.

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. Analternative desired effect for the co-agonist analogs of the presentdisclosure would include treating hyperglycemia, e.g., as measured by achange in blood glucose level closer to normal, or inducing weightloss/preventing weight gain, e.g., as measured by reduction in bodyweight, or preventing or reducing an increase in body weight, ornormalizing body fat distribution. The amount that is “effective” willvary from subject to subject, depending on the age and general conditionof the individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment. As used herein, the term “purified” doesnot require absolute purity; rather, it is intended as a relativedefinition. The term “purified polypeptide” is used herein to describe apolypeptide which has been separated from other compounds including, butnot limited to nucleic acid molecules, lipids and carbohydrates.

The term “isolated” requires that the referenced material be removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotidepresent in a living animal is not isolated, but the same polynucleotide,separated from some or all of the coexisting materials in the naturalsystem, is isolated.

As used herein, the term “peptide” encompasses a sequence of 3 or moreamino acids and typically less than 50 amino acids, wherein the aminoacids are naturally occurring or non-naturally occurring amino acids.Non-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein.

As used herein, the terms “polypeptide” and “protein” are terms that areused interchangeably to refer to a polymer of amino acids, withoutregard to the length of the polymer. Typically, polypeptides andproteins have a polymer length that is greater than that of “peptides.”

A “glucagon peptide” as used herein includes any peptide comprising,either the amino acid sequence of SEQ ID NO: 1, or any analog of theamino acid sequence of SEQ ID NO: 1, including amino acid substitutions,additions, deletions or post translational modifications (e.g.,methylation, acylation, ubiquitination, intramolecular covalent bondingsuch as lactam bridge formation, PEGylation, and the like) of thepeptide, wherein the analog stimulates glucagon or GLP-1 receptoractivity, e.g., as measured by cAMP production using the assay describedin Example 14.

The term “glucagon agonist” refers to a complex comprising a glucagonpeptide that stimulates glucagon receptor activity, e.g., as measured bycAMP production using the assay described in Example 14.

As used herein a “glucagon agonist analog” is a glucagon peptidecomprising a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQID NO: 15, or an analog of such a sequence that has been modified toinclude one or more conservative amino acid substitutions at one or moreof positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28or 29.

As used herein an amino acid “modification” refers to a substitution,addition or deletion of an amino acid, and includes substitution with oraddition of any of the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids. Throughoutthe application, all references to a particular amino acid position bynumber (e.g. position 28) refer to the amino acid at that position innative glucagon (SEQ ID NO:1) or the corresponding amino acid positionin any analogs thereof. For example, a reference herein to “position 28”would mean the corresponding position 27 for a glucagon analog in whichthe first amino acid of SEQ ID NO: 1 has been deleted. Similarly, areference herein to “position 28” would mean the corresponding position29 for a glucagon analog in which one amino acid has been added beforethe N-terminus of SEQ ID NO: 1. Commercial sources of atypical aminoacids include Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc. (Miami,Fla.), and Genzyme Pharmaceuticals (Cambridge, Mass.). Atypical aminoacids may be purchased from commercial suppliers, synthesized de novo,or chemically modified or derivatized from other amino acids.

As used herein a “glucagon co-agonist” is a glucagon peptide thatexhibits activity at the glucagon receptor of at least about 10% toabout 500% or more relative to native glucagon and also exhibitsactivity at the GLP-1 receptor of about at least 10% to about 200% ormore relative to native GLP-1.

As used herein a “glucagon/GLP-1 co-agonist molecule” is a molecule thatexhibits activity at the glucagon receptor of at least about 10%relative to native glucagon and also exhibits activity at the GLP-1receptor of at least about 10% relative to native GLP-1.

As used herein the term “native glucagon” refers to a peptide consistingof the sequence of SEQ ID NO: 1, and the term “native GLP-1” is ageneric term that designates GLP-1 (7-36)amide (consisting of thesequence of SEQ ID NO: 52), GLP-1(7-37)acid (consisting of the sequenceof SEQ ID NO: 50) or a mixture of those two compounds. As used herein, ageneral reference to “glucagon” or “GLP-1” in the absence of any furtherdesignation is intended to mean native glucagon or native GLP-1,respectively.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol chain” or “PEGchain”, refers to mixtures of condensation polymers of ethylene oxideand water, in a branched or straight chain, represented by the generalformula H(OCH₂CH₂)_(n)OH, wherein n is at least 9. Absent any furthercharacterization, the term is intended to include polymers of ethyleneglycol with an average total molecular weight selected from the range of500 to 40,000 Daltons. “polyethylene glycol chain” or “PEG chain” isused in combination with a numeric suffix to indicate the approximateaverage molecular weight thereof. For example, PEG-5,000 refers topolyethylene glycol chain having a total molecular weight average ofabout 5,000.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol chain to the compound. A “pegylated glucagon peptide” is aglucagon peptide that has a PEG chain covalently bound to the glucagonpeptide.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid chain comprising an amide group in place of the terminalcarboxylic acid is intended to be encompassed by an amino acid sequencedesignating the standard amino acids.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein a “dimer” is a complex comprising two subunits covalentlybound to one another via a linker. The term dimer, when used absent anyqualifying language, encompasses both homodimers and heterodimers. Ahomodimer comprises two identical subunits, whereas a heterodimercomprises two subunits that differ, although the two subunits aresubstantially similar to one another.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that is negatively charged (i.e.,de-protonated) or positively charged (i.e., protonated) in aqueoussolution at physiological pH. For example negatively charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positively charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety, including for example, a carboxylicacid or sulfonic acid group.

The term “alkyl” refers to a linear or branched hydrocarbon containingthe indicated number of carbon atoms. Exemplary alkyls include methyl,ethyl, and linear propyl groups.

The term “heteroalkyl” refers to a linear or branched hydrocarboncontaining the indicated number of carbon atoms and at least oneheteroatom in the backbone of the structure. Suitable heteroatoms forpurposes herein include but are not limited to N, S, and O.

EMBODIMENTS

The invention provides glucagon peptides with increased or decreasedactivity at the glucagon receptor, or the GLP-1 receptor, or at bothreceptors. The invention also provides glucagon peptides with alteredselectivity for the glucagon receptor versus the GLP-1 receptor.

Increased activity at the glucagon receptor is provided by an amino acidmodification at position 16 of native glucagon (SEQ ID NO: 1) asdescribed herein.

Maintained or increased activity at the glucagon receptor is alsoprovided by an amino acid modification at position 3 of native glucagonwith a glutamine analog (e.g. (Dab(Ac)).

Reduced activity at the glucagon receptor is provided, e.g., bysubstitution of the amino acid at position 3 with an acidic, basic, orhydrophobic amino acid as described herein.

Increased activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester.

Increased activity at the GLP-1 receptor is provided by modificationsthat stabilize the alpha helix in the C-terminal portion of glucagon(e.g. around residues 12-29). In some embodiments, such modificationspermit formation of an intramolecular bridge between the side chains oftwo amino acids that are separated by three intervening amino acids, forexample, positions 12 and 16, or 16 and 20, or and 24, as describedherein. In other embodiments, such modifications include insertion orsubstitution modifications that introduce one or more α,α-disubstitutedamino acids, e.g. AIB at one or more of positions 16, 20, 21 or 24.

Increased activity at the GLP-1 and glucagon receptors for peptideslacking an intramolecular bridge, e.g., a covalent intramolecularbridge, is provided by covalently attaching an acyl or alkyl group tothe side chain of the amino acid at position 10 of the peptide, whereinthe acyl or alkyl group is non-native to the amino acid at position 10.Further increased activity at the GLP-1 and glucagon receptors for suchpeptides lacking an intramolecular bridge, e.g., a covalentintramolecular bridge, may be achieved by incorporating a spacer betweenthe acyl or alkyl group and the side chain of the amino acid at position10. Suitable spacers are described herein and include, but not limitedto spacers that are 3 to 10 atoms in length.

Increased activity at the GLP-1 receptor is provided by an amino acidmodification at position 20 as described herein.

Increased activity at the GLP-1 receptor is provided in glucagon analogscomprising the C-terminal extension of SEQ ID NO: 26. GLP-1 activity insuch analogs comprising SEQ ID NO: 26 can be further increased bymodifying the amino acid at position 18, 28 or 29, or at position 18 and29, as described herein.

Restoration of glucagon activity which has been reduced by amino acidmodifications at positions 1 and 2 is provided by a covalent bondbetween the side chains of two amino acids that are separated by threeintervening amino acids, for example, positions 12 and 16, or 16 and 20,or 20 and 24, as described herein.

A further modest increase in GLP-1 potency is provided by modifying theamino acid at position 10 to be Trp.

Any of the modifications described above which increase or decreaseglucagon receptor activity and which increase GLP-1 receptor activitycan be applied individually or in combination. Any of the modificationsdescribed above can also be combined with other modifications thatconfer other desirable properties, such as increased solubility and/orstability and/or duration of action. Alternatively, any of themodifications described above can be combined with other modificationsthat do not substantially affect solubility or stability or activity.Exemplary modifications include but are not limited to:

(A) Improving solubility, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. Such a charged aminoacid can be introduced by substituting a native amino acid with acharged amino acid, e.g. at positions 28 or 29, or alternatively byadding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged. Such modificationsincrease solubility, e.g. provide at least 2-fold, 5-fold, 10-fold,15-fold, 25-fold, 30-fold or greater solubility relative to nativeglucagon at a given pH between about 5.5 and 8, e.g., pH 7, whenmeasured after 24 hours at 25° C.

(B) Increasing solubility and duration of action or half-life incirculation by addition of a hydrophilic moiety such as a polyethyleneglycol chain, as described herein, e.g. at position 16, 17, 20, 21, 24or 29, or at the C-terminal amino acid of the peptide.

(C) Increasing, by modification of the aspartic acid at position 15, forexample, by deletion or substitution with glutamic acid, homoglutamicacid, cysteic acid or homocysteic acid. Such modifications can reducedegradation or cleavage at a pH within the range of 5.5 to 8, forexample, retaining at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% ofthe original peptide after 24 hours at 25° C.

(D) Increasing stability by modification of the methionine at position27, for example, by substitution with leucine or norleucine. Suchmodifications can reduce oxidative degradation. Stability can also beincreased by modification of the Gln at position 20 or 24, e.g. bysubstitution with Ala, Ser, Thr, or AIB. Such modifications can reducedegradation that occurs through deamidation of Gln. Stability can beincreased by modification of Asp at position 21, e.g. by substitutionwith Glu. Such modifications can reduce degradation that occurs throughdehydration of Asp to form a cyclic succinimide intermediate followed byisomerization to iso-aspartate.

(E) Increasing resistance to dipeptidyl peptidase IV (DPP IV) cleavageby modification of the amino acid at position 1 or 2 as describedherein.

(F) Conservative or non-conservative substitutions, additions ordeletions that do not affect activity, for example, conservativesubstitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14,16, 17, 18, 19, 20, 21, 24, 27, 28 or 29; deletions at one or more ofpositions 27, 28 or 29; or a deletion of amino acid 29 optionallycombined with a C-terminal amide or ester in place of the C-terminalcarboxylic acid group;

(G) Adding C-terminal extensions as described herein;

(H) Increasing half-life in circulation and/or extending the duration ofaction and/or delaying the onset of action, for example, throughacylation or alkylation of the glucagon peptide, as described herein;

(I) Homodimerization or heterodimerization as described herein.

In exemplary embodiments, the glucagon peptide may comprise a total of1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to9, or up to 10 amino acid modifications relative to the native glucagonsequence.

Other modifications include substitution of His at position 1 with alarge, aromatic amino acid (e.g., Tyr, Phe, Trp or amino-Phe);

Ser at position 2 with Ala;

substitution of Tyr at position 10 with Val or Phe;

substitution of Lys at position 12 with Arg;

substitution of Asp at position 15 with Glu;

substitution of Ser at position 16 with Thr or AIB.

One embodiment disclosed herein is directed to a glucagon agonist thathas been modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1) to enhance the peptide's potency at the glucagonreceptor. Surprisingly, applicants have discovered that the normallyoccurring serine at position 16 of native glucagon (SEQ ID NO: 1) can besubstituted with select acidic amino acids to enhance the potency ofglucagon, in terms of its ability to stimulate cAMP synthesis in avalidated in vitro model assay (see Example 14). More particularly, thissubstitution enhances the potency of the analog at least 2-fold, 4-fold,5-fold, and up to 10-fold greater at the glucagon receptor. Thissubstitution also enhances the analog's activity at the GLP-1 receptorat least 5-fold, 10-fold, or 15-fold relative to native glucagon, butselectivity is maintained for the glucagon receptor over the GLP-1receptor.

In accordance with one embodiment the serine residue at position 16 ofnative glucagon is substituted with an amino acid selected from thegroup consisting of glutamic acid, glutamine, homoglutamic acid,homocysteic acid, threonine or glycine. In accordance with oneembodiment the serine residue at position 16 of native glucagon issubstituted with an amino acid selected from the group consisting ofglutamic acid, glutamine, homoglutamic acid and homocysteic acid, and inone embodiment the serine residue is substituted with glutamic acid. Inone embodiment the glucagon peptide having enhanced specificity for theglucagon receptor comprises the peptide of SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10 or a glucagon agonist analog thereof, wherein the carboxyterminal amino acid retains its native carboxylic acid group. Inaccordance with one embodiment a glucagon agonist comprising thesequence ofNH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH(SEQ ID NO: 10) is provided, wherein the peptide exhibits approximatelyfivefold enhanced potency at the glucagon receptor, relative to nativeglucagon as measured by the in vitro cAMP assay of Example 14.

Hydrophilic Moieties

The glucagon peptides of the present invention can be further modifiedto improve the peptide's solubility and stability in aqueous solutionsat physiological pH, while retaining the high biological activityrelative to native glucagon. Hydrophilic moieties such as PEG groups canbe attached to the glucagon peptides under any suitable conditions usedto react a protein with an activated polymer molecule. Any means knownin the art can be used, including via acylation, reductive alkylation,Michael addition, thiol alkylation or other chemoselectiveconjugation/ligation methods through a reactive group on the PEG moiety(e.g., an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido orhydrazino group) to a reactive group on the target compound (e.g., analdehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazinogroup). Activating groups which can be used to link the water solublepolymer to one or more proteins include without limitation sulfone,maleimide, sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane,5-pyridyl, and alpha-halogenated acyl group (e.g., alpha-iodo aceticacid, alpha-bromoacetic acid, alpha-chloroacetic acid). If attached tothe peptide by reductive alkylation, the polymer selected should have asingle reactive aldehyde so that the degree of polymerization iscontrolled. See, for example, Kinstler et al., Adv. Drug. Delivery Rev.54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476(2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

In a specific aspect of the invention, an amino acid residue on theglucagon peptide having a thiol is modified with a hydrophilic moietysuch as PEG. In some embodiments, the thiol is modified withmaleimide-activated PEG in a Michael addition reaction to result in aPEGylated peptide comprising the thioether linkage shown below:

In some embodiments, the thiol is modified with a haloacetyl-activatedPEG in a nucleophilic substitution reaction to result in a PEGylatedpeptide comprising the thioether linkage shown below:

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.Dextrans are polysaccharide polymers of glucose subunits, predominantlylinked by α1-6 linkages. Dextran is available in many molecular weightranges, e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. Linear or branchedpolymers are contemplated. Resulting preparations of conjugates may beessentially monodisperse or polydisperse, and may have about 0.5, 0.7,1, 1.2, 1.5 or 2 polymer moieties per peptide.

In accordance with one embodiment, introduction of hydrophilic groups atpositions 17, 21, and 24 of the peptide of SEQ ID NO: 9 or SEQ ID NO: 10are anticipated to improve the solubility and stability of the highpotency glucagon analog in solutions having a physiological pH.Introduction of such groups also increases duration of action, e.g. asmeasured by a prolonged half-life in circulation. Suitable hydrophilicmoieties include any water soluble polymers known in the art, includingPEG, homo- or co-polymers of PEG, a monomethyl-substituted polymer ofPEG (mPEG), or polyoxyethylene glycerol (POG). In accordance with oneembodiment the hydrophilic group comprises a polyethylene (PEG) chain.More particularly, in one embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 6 or SEQ ID NO: 7 wherein a PEG chain iscovalently linked to the side chains of amino acids present at positions21 and 24 of the glucagon peptide and the carboxy terminal amino acid ofthe peptide has the carboxylic acid group.

Conjugates

The present disclosure also encompasses other conjugates in whichglucagon peptides of the invention are linked, optionally via covalentbonding and optionally via a linker, to a conjugate moiety. Linkage canbe accomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

The peptide can be linked to conjugate moieties via direct covalentlinkage by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of these targeted aminoacids. Reactive groups on the peptide or conjugate moiety include, e.g.,an aldehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazinogroup. Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art. Alternatively, the conjugatemoieties can be linked to the peptide indirectly through intermediatecarriers, such as polysaccharide or polypeptide carriers. Examples ofpolysaccharide carriers include aminodextran. Examples of suitablepolypeptide carriers include polylysine, polyglutamic acid, polyasparticacid, co-polymers thereof, and mixed polymers of these amino acids andothers, e.g., serines, to confer desirable solubility properties on theresultant loaded carrier.

Cysteinyl residues are most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid, chloroacetamideto give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues also are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagine or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the peptide. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Exemplary conjugate moieties that can be linked to any of the glucagonpeptides described herein include but are not limited to a heterologouspeptide or polypeptide (including for example, a plasma protein), atargeting agent, an immunoglobulin or portion thereof (e.g. variableregion, CDR, or Fc region), a diagnostic label such as a radioisotope,fluorophore or enzymatic label, a polymer including water solublepolymers, or other therapeutic or diagnostic agents. In one embodiment aconjugate is provided comprising a glucagon peptide of the presentinvention and a plasma protein, wherein the plasma protein is selectedform the group consisting of albumin, transferin, fibrinogen andglobulins. In one embodiment the plasma protein moiety of the conjugateis albumin or transferin.

In some embodiments, the linker comprises a chain of atoms from 1 toabout 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atomsare all carbon atoms. In some embodiments, the chain atoms in thebackbone of the linker are selected from the group consisting of C, O,N, and S. Chain atoms and linkers may be selected according to theirexpected solubility (hydrophilicity) so as to provide a more solubleconjugate. In some embodiments, the linker provides a functional groupthat is subject to cleavage by an enzyme or other catalyst or hydrolyticconditions found in the target tissue or organ or cell. In someembodiments, the length of the linker is long enough to reduce thepotential for steric hindrance. If the linker is a covalent bond or apeptidyl bond and the conjugate is a polypeptide, the entire conjugatecan be a fusion protein. Such peptidyl linkers may be any length.Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50,3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length. Such fusionproteins may alternatively be produced by recombinant geneticengineering methods known to one of ordinary skill in the art.

As noted above, in some embodiments, the glucagon peptides areconjugated, e.g., fused to an immunoglobulin or portion thereof (e.g.variable region, CDR, or Fc region). Known types of immunoglobulins (Ig)include IgG, IgA, IgE, IgD or IgM. The Fc region is a C-terminal regionof an Ig heavy chain, which is responsible for binding to Fc receptorsthat carry out activities such as recycling (which results in prolongedhalf-life), antibody dependent cell-mediated cytotoxicity (ADCC), andcomplement dependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J Immunol. 175:6694-701, 2005) Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Fusion Protein and Terminal Extension

The present disclosure also encompasses glucagon fusion peptides orproteins wherein a second peptide or polypeptide has been fused to aterminus, e.g., the carboxy terminus of the glucagon peptide. Moreparticularly, the fusion glucagon peptide may comprise a glucagonagonist of SEQ ID NO: 55, SEQ ID NO: 9 or SEQ

ID NO: 10 further comprising an amino acid sequence of SEQ ID NO: 26(GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked toamino acid 29 of the glucagon peptide. In one embodiment the amino acidsequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQID NO: 28 (KRNR) is bound to amino acid 29 of the glucagon peptidethrough a peptide bond. Applicants have discovered that in glucagonfusion peptides comprising the C-terminal extension peptide of Exendin-4(e.g., SEQ ID NO: 26 or SEQ ID NO: 29), substitution of the nativethreonine residue at position 29 with glycine dramatically increasesGLP-1 receptor activity. This amino acid substitution can be used inconjunction with other modifications disclosed herein to enhance theaffinity of the glucagon analogs for the GLP-1 receptor. For example,the T29G substitution can be combined with the S16E and N20K amino acidsubstitutions, optionally with a lactam bridge between amino acids 16and 20, and optionally with addition of a PEG chain as described herein.In one embodiment a glucagon/GLP-1 receptor co-agonist is provided,comprising the sequence of SEQ ID NO: 64. In one embodiment the glucagonpeptide portion of the glucagon fusion peptide is selected from thegroup consisting of SEQ ID NO: 55, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5 wherein a PEG chain, when present at positions17, 21, 24, or the C-terminal amino acid, or at both 21 and 24, isselected from the range of 500 to 40,000 Daltons. More particularly, inone embodiment the glucagon peptide segment is selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 63, wherein thePEG chain is selected from the range of 500 to 5,000. In one embodimentthe glucagon peptide is a fusion peptide comprising the sequence of SEQID NO: 55 and SEQ ID NO: 65 wherein the peptide of SEQ ID NO: 65 islinked to the carboxy terminus of SEQ ID NO: 55.

Charge Neutral C-Terminus

In accordance with one embodiment, an additional chemical modificationof the glucagon peptide of SEQ ID NO: 10 bestows increased GLP-1receptor potency to a point where the relative activity at the glucagonand GLP-1 receptors is virtually equivalent. Accordingly, in oneembodiment a glucagon/GLP-1 receptor co-agonist is provided wherein theterminal amino acid of the glucagon peptides of the present inventionhave an amide group in place of the carboxylic acid group that ispresent on the native amino acid. The relative activity of the glucagonanalog at the respective glucagon and GLP-1 receptors can be adjusted byfurther modifications to the glucagon peptide to produce analogsdemonstrating about 40% to about 500% or more of the activity of nativeglucagon at the glucagon receptor and about 20% to about 200% or more ofthe activity of native GLP-1 at the GLP-1 receptor, e.g. 50-fold,100-fold or more increase relative to the normal activity of glucagon atthe GLP-1 receptor. In some embodiments, the glucagon peptides describedherein exhibit up to about 100%, 1000%, 10,000%, 100,000%, or 1,000,000%of the activity of native glucagon at the glucagon receptor. In someembodiments, the glucagon peptides described herein exhibit up to about100%, 1000%, 10,000%, 100,000%, or 1,000,000% of the activity of nativeGLP-1 at the GLP-1 receptor.

Stabilization of the Alpha Helix/Intramolecular Bridges

In a further embodiment glucagon analogs are provided that exhibitenhanced GLP-1 receptor agonist activity wherein an intramolecularbridge is formed between two amino acid side chains to stabilize thethree dimensional structure of the carboxy terminus of the peptide. Thetwo amino acid side chains can be linked to one another throughnon-covalent bonds, e.g., hydrogen-bonding, ionic interactions, such asthe formation of salt bridges, or by covalent bonds. When the two aminoacid side chains are linked to one another through one or more covalentbonds, the peptide may be considered herein as comprising a covlentintramolecular bridge. When the two amino acid side chains are linked toone another through non-covalent bonds, e.g., hydrogen bonds, ionicinteractions, the peptide may be considered herein as comprising anon-covalent intramolecular bridge.

In some embodiments, the intramolecular bridge is formed between twoamino acids that are 3 amino acids apart, e.g., amino acids at positionsi and i+4, wherein i is any integer between 12 and 25 (e.g., 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25). More particularly, theside chains of the amino acid pairs 12 and 16, 16 and 20, 20 and 24 or24 and 28 (amino acid pairs in which i=12, 16, 20, or 24) are linked toone another and thus stabilize the glucagon alpha helix. Alternatively,i can be 17.

In some specific embodiments, wherein the amino acids at positions i andi+4 are joined by an intramolecular bridge, the size of the linker isabout 8 atoms, or about 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are two amino acids apart, e.g., amino acids atpositions j and j+3, wherein j is any integer between 12 and 26 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26). In somespecific embodiments, j is 17.

In some specific embodiments, wherein amino acids at positions j and j+3are joined by an intramolecular bridge, the size of the linker is about6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are 6 amino acids apart, e.g., amino acids atpositions k and k+7, wherein k is any integer between 12 and 22 (e.g.,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22). In some specificembodiments, k is 12, 13, or 17. In an exemplary embodiment, k is 17.

Examples of amino acid pairings that are capable of covalently bondingto form a six-atom linking bridge include Orn and Asp, Glu and an aminoacid of Formula I, wherein n is 2, and homoglutamic acid and an aminoacid of Formula I, wherein n is 1, wherein Formula I is:

Examples of amino acid pairing that are capable of covalently bonding toform a seven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modified derivatives, that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect. Even without covalentlinkage, the amino acid pairings described above or similar pairingsthat one of ordinary skill in the art can envision may also provideadded stability to the alpha-helix through non-covalent bonds, forexample, through formation of salt bridges or hydrogen-bondinginteractions.

Further exemplary embodiments include the following pairings, optionallywith a lactam bridge: Glu at position 12 with Lys at position 16; nativeLys at position 12 with Glu at position 16; Glu at position 16 with Lysat position 20; Lys at position 16 with Glu at position 20; Glu atposition 20 with Lys at position 24; Lys at position 20 with Glu atposition 24; Glu at position 24 with Lys at position 28; Lys at position24 with Glu at position 28.

In accordance with one embodiment a glucagon analog is provided thatexhibits glucagon/GLP-1 receptor co-agonist activity wherein the analogcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 11, 47, 48 and 49. In one embodiment the side chains arecovalently bound to one another, and in one embodiment the two aminoacids are bound to one another to form a lactam ring. The size of thelactam ring can vary depending on the length of the amino acid sidechains, and in one embodiment the lactam is formed by linking the sidechains of a lysine amino acid to a glutamic acid side chain.

The order of the amide bond in the lactam ring can be reversed (e.g., alactam ring can be formed between the side chains of a Lys12 and a Glu16or alternatively between a Glu 12 and a Lys16). In accordance with oneembodiment a glucagon analog of SEQ ID NO: 45 is provided wherein atleast one lactam ring is formed between the side chains of an amino acidpair selected from the group consisting of amino acid pairs 12 and 16,16 and 20, 20 and 24 or 24 and 28. In one embodiment a glucagon/GLP-1receptor co-agonist is provided wherein the co-agonist comprises aglucagon peptide analog of SEQ ID NO: 20 wherein the peptide comprisesan intramolecular lactam bridge formed between amino acid positions 12and 16 or between amino acid positions 16 and 20. In one embodiment aglucagon/GLP-1 receptor co-agonist is provided comprising the sequenceof SEQ ID NO: 20, wherein an intramolecular lactam bridge is formedbetween amino acid positions 12 and 16, between amino acid positions 16and 20, or between amino acid positions 20 and 24 and the amino acid atposition 29 is glycine, wherein the sequence of SEQ ID NO: 29 is linkedto the C-terminal amino acid of SEQ ID NO: 20. In a further embodimentthe amino acid at position 28 is aspartic acid.

Intramolecular bridges other than a lactam bridge can be used tostabilize the alpha helix of the glucagon analog peptides. In oneembodiment, the intramolecular bridge is a hydrophobic bridge. In thisinstance, the intramolecular bridge optionally is between the sidechains of two amino acids that are part of the hydrophobic face of thealpha helix of the glucagon analog peptide. For example, one of theamino acids joined by the hydrophobic bridge can be the amino acid atposition 10, 14, and 18.

In one specific aspect, olefin metathesis is used to cross-link one ortwo turns of the alpha helix of the glucagon peptide using anall-hydrocarbon cross-linking system. The glucagon peptide in thisinstance can comprise α-methylated amino acids bearing olefinic sidechains of varying length and configured with either R or Sstereochemistry at the i and i+4 or i+7 positions. For example, theolefinic side can can comprise (CH₂)n, wherein n is any integer between1 to 6. In one embodiment, n is 3 for a cross-link length of 8 atoms.Suitable methods of forming such intramolecular bridges are described inthe art. See, for example, Schafineister et al., J. Am. Chem. Soc. 122:5891-5892 (2000) and Walensky et al., Science 305: 1466-1470 (2004).Alternatively, the glucagon peptide can comprise O-allyl Ser residueslocated on adjacent helical turns, which are bridged together viaruthenium-catalyzed ring closing metathesis. Such procedures ofcross-linking are described in, for example, Blackwell et al., Angew,Chem., Int. Ed. 37: 3281-3284 (1998).

In another specific aspect, use of the unnatural thio-dialanine aminoacid, lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α,ω-diaminoalkane tethers, e.g., 1,4-diaminopropaneand 1,5-diaminopentane) between two Glu residues at positions i and i+7are used to stabilize the alpha helix of the glucagon peptide. Suchtethers lead to the formation of a bridge 9-atoms or more in length,depending on the length of the diaminoalkane tether. Suitable methods ofproducing peptides cross-linked with such tethers are described in theart. See, for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460(1997).

In yet another embodiment of the invention, a disulfide bridge is usedto cross-link one or two turns of the alpha helix of the glucagonpeptide. Alternatively, a modified disulfide bridge in which one or bothsulfur atoms are replaced by a methylene group resulting in an isostericmacrocyclization is used to stabilize the alpha helix of the glucagonpeptide. Suitable methods of modifying peptides with disulfide bridgesor sulfur-based cyclization are described in, for example, Jackson etal., J. Am. Chem. Soc. 113: 9391-9392 (1991) and Rudinger and Jost,Experientia 20: 570-571 (1964).

In yet another embodiment, the alpha helix of the glucagon peptide isstabilized via the binding of metal atom by two His residues or a Hisand Cys pair positioned at i and i+4. The metal atom can be, forexample, Ru(III), Cu(II), Zn(II), or Cd(II). Such methods of metalbinding-based alpha helix stabilization are known in the art. See, forexample, Andrews and Tabor, Tetrahedron 55: 11711-11743 (1999); Ghadiriet al., J. Am. Chem. Soc. 112: 1630-1632 (1990); and Ghadiri et al., J.Am. Chem. Soc. 119: 9063-9064 (1997).

The alpha helix of the glucagon peptide can alternatively be stabilizedthrough other means of peptide cyclizing, which means are reviewed inDavies, J. Peptide. Sci. 9: 471-501 (2003). The alpha helix can bestabilized via the formation of an amide bridge, thioether bridge,thioester bridge, urea bridge, carbamate bridge, sulfonamide bridge, andthe like. For example, a thioester bridge can be formed between theC-terminus and the side chain of a Cys residue. Alternatively, athioester can be formed via side chains of amino acids having a thiol(Cys) and a carboxylic acid (e.g., Asp, Glu). In another method, across-linking agent, such as a dicarboxylic acid, e.g. suberic acid(octanedioic acid), etc. can introduce a link between two functionalgroups of an amino acid side chain, such as a free amino, hydroxyl,thiol group, and combinations thereof.

In accordance with one embodiment, the alpha helix of the glucagonpeptide is stabilized through the incorporation of hydrophobic aminoacids at positions i and i+4. For instance, i can be Tyr and i+4 can beeither Val or Leu; i can be Phe and i+4 can be Cys or Met; I can be Cysand i+4 can be Met; or i can be Phe and i+4 can be Ile. It should beunderstood that, for purposes herein, the above amino acid pairings canbe reversed, such that the indicated amino acid at position i couldalternatively be located at i+4, while the i+4 amino acid can be locatedat the i position.

In accordance with yet another embodiment of the invention, the glucagonpeptide with enhanced GLP-1 activity comprises (a) one or moresubstitutions within amino acid positions 12-29 with anα,α-disubstituted amino acid and optionally, (b) a C-terminal amide. Insome aspects, it is to be appreciated that such glucagon peptidesspecifically lack an intramolecular bridge, e.g., a covalentintramolecular bridge, that stabilizes the alpha-helix in the C-terminalportion of glucagon (around positions 12-29). In some embodiments, one,two, three, four or more of positions 16, 17, 18, 19, 20, 21, 24 or 29of glucagon is substituted with an α,α-disubstituted amino acid, e.g.,amino iso-butyric acid (AIB), an amino acid disubstituted with the sameor a different group selected from methyl, ethyl, propyl, and n-butyl,or with a cyclooctane or cycloheptane (e.g.,1-aminocyclooctane-1-carboxylic acid). For example, substitution ofposition 16 with AIB enhances GLP-1 activity, in the absence of anintramolecular bridge, e.g., a non-covalent intramolecular bridge (e.g.,a salt bridge) or a covalent intramolecular bridge (e.g., a lactam). Insome embodiments, one, two, three or more of positions 16, 20, 21 or 24are substituted with AIB. Such a glucagon peptide may further compriseone or more of the other modifications described herein, including, butnot limited to, acylation, alkylation, pegylation, deletion of 1-2 aminoacids at the C-terminus, addition of and/or substitution with chargedamino acids at the C-terminus, replacement of the C-terminal carboxylatewith an amide, addition of a C-terminal extension, and conservativeand/or non-conservative amino acid substitutions, such as substitutionof Met at position 27 with Leu or Nle, substitution of Asp at position15 with Glu (or like amino acid), substitution at position 1 and/or 2with amino acids which achieve DPP-IV protease resistance, substitutionof Ser at position 2 with Ala, substitution of Tyr at position 10 withVal or Phe, substitution of Lys at position 12 with Arg, substitution ofSer at position 16 with Thr or AIB, substitution of Gln at position 20and/or 24 with Asp, Glu, or AIB, substitution of Ser at position 16 withGlu or Thr, Arg at position 18 with Ala, Gln at position 20 with Lys,Asp at position 21 with Glu, and Gln at position 24 with Asn or Cys. Insome embodiments, the foregoing glucagon peptide comprises a Gln or Glyat position 29 or addition of a C-terminal extension, e.g., GGPSSGAPPPS(SEQ ID NO: 26) C-terminal to the amino acid at position 28. In aspecific aspect, the glucagon peptide comprises one or more of an amidegroup in place of the C-terminal carboxylate, an acyl group, e.g., a C16fatty acid, and a hydrophilic moiety, e.g., a polyethylene glycol (PEG).

Also, in another specific aspect, the glucagon peptide comprises theamino acid sequence of any of SEQ ID NOs: 1-25, 30-64, and 66-555comprising no more than ten modifications relative to SEQ ID NO: 1 andcomprising one or more amino acid substitutions with AIB at positions16, 20, 21, and/or 24, wherein the peptide lacks an intramolecularbridge, e.g., a covalent intramolecular bridge, between the side chainsof two amino acids of the peptide. Accordingly, in a more specificaspect, the glucagon peptide comprises the amino acid sequence of any ofSEQ ID NOs: 556-561.

In accordance with some embodiments, the glucagon peptide lacking anintramolecular bridge comprises one or more substitutions within aminoacid positions 12-29 with an α,α-disubstituted amino acid and an acyl oralkyl group covalently attached to the side chain of the amino acid atposition 10 of the glucagon peptide. In specific embodiments, the acylor alkyl group is not naturally occurring on an amino acid. In certainaspects, the acyl or alkyl group is non-native to the amion acid atposition 10. In exemplary embodiments, the glucagon peptide lacking anintramolecular bridge comprises the amino acid sequence of any of SEQ IDNOs: 556-561 and an acyl or alkyl group covalently attached to the sidechain of the amino acid at position 10 of the glucagon peptide. Suchacylated or alkylated glucagon peptides lacking an intramolecular bridgeexhibit enhanced activity at the GLP-1 and glucagon receptors ascompared to the non-acylated counterpart peptides. Further enhancementin activity at the GLP-1 and glucagon receptors can be achieved by theacylated glucagon peptides lacking an intramolecular bridge byincorporating a spacer between the acyl or alkyl group and the sidechain of the amino acid at position 10 of the peptide. Acylation andalkylation, with or without incorporating spacers, are further describedherein.

Modification at Position 1

In accordance with one embodiment of the invention, the glucagon peptidewith enhanced GLP-1 activity comprises (a) an amino acid substitution ofHis at position 1 with a large, aromatic amino acid and (b) anintramolecular bridge that stabilizes that alpha-helix in the C-terminalportion of the molecule (e.g. around positions 12-29). In a specificembodiment, the amino acid at position 1 is Tyr, Phe, Trp, amino-Phe,nitro-Phe, chloro-Phe, sulfo-Phe, 4-pyridyl-Ala, methyl-Tyr, or 3-aminoTyr. In a specific aspect, the intramolecular bridge is between the sidechains of two amino acids that are separated by three intervening aminoacids, i.e., between the side chains of amino acids i and i+4. In someembodiments, the intramolecular bridge is a lactam bridge. In a morespecific embodiment of the invention, the glucagon peptide comprises alarge, aromatic amino acid at position 1 and a lactam bridge between theamino acids at positions 16 and 20 of the peptide. Such a glucagonpeptide may further comprise one or more (e.g., two, three, four, fiveor more) of the other modifications described herein. For example, theglucagon peptide can comprise an amide in place of the C-terminalcarboxylate. Accordingly, in one embodiment, the glucagon peptidecomprises that amino acid sequence of SEQ ID NO: 555.

Acylation

In accordance with one embodiment, the glucagon peptide comprises anacyl group, e.g., an acyl group which is non-native to anaturally-occurring amino acid. The acyl group causes the peptide tohave one or more of (i) a prolonged half-life in circulation, (ii) adelayed onset of action, (iii) an extended duration of action, (iv) animproved resistance to proteases, such as DPP-IV, and (v) increasedpotency at the GLP-1 receptor, GIP receptor, and/or glucagon receptor.As shown herein, acylated glucagon peptides do not exhibit decreasedactivity at the glucagon receptor, GIP receptor, and/or, GLP-1 receptorin comparison to the corresponding unacylated glucagon peptide. Rather,in some instances, acylated glucagon peptides actually exhibit increasedactivity at the GLP-1 receptor, GIP receptor, and/or glucagon receptor.Accordingly, the potency of the acylated analogs is comparable to theunacylated versions of the glucagon co-agonist analogs, if not enhanced.

In accordance with one embodiment, the glucagon peptide is modified tocomprise an acyl group which is attached to the glucagon peptide via anester, thioester, or amide linkage for purposes of prolonging half-lifein circulation and/or delaying the onset of and/or extending theduration of action and/or improving resistance to proteases such asDPP-IV.

Acylation can be carried out at any position within the glucagonpeptide, including any of positions 1-29, a position within a C-terminalextension, or the C-terminal amino acid, provided that glucagon and/orGLP-1 activity and/or GIP activity is retained, if not enhanced.Nonlimiting examples include positions 5, 7, 10, 11, 12, 13, 14, 16, 17,18, 19, 20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 40, 41, 42, or 43(according to the numbering of the amino acids of SEQ ID NO: 1). Inspecific embodiments, acylation occurs at position 10 or 40 of theglucagon peptide and the glucagon peptide lacks an intramolecularbridge, e.g., a covalent intramolecular bridge (e.g., a lactam bridge)and comprises a C-terminal extension. Such acylated peptides lacking anintramolecular bridge and comprising a C-terminal extension demonstrateenhanced activity at the GLP-1, GIP, and glucagon receptors as comparedto the corresponding non-acylated peptides. Accordingly, the position atwhich acylation occurs can alter the overall activity profile of theglucagon analog.

Glucagon peptides may be acylated at the same amino acid position wherea hydrophilic moiety is linked, or at a different amino acid position.Nonlimiting examples include acylation at position 10 or 40 andpegylation at one or more positions in the C-terminal portion of theglucagon peptide, e.g., position 24, 28, 29, or 40 within a C-terminalextension, or at the C-terminus (e.g., through adding a C-terminal Cys).

The acyl group can be covalently linked directly to an amino acid of theglucagon peptide, or indirectly to an amino acid of the glucagon peptidevia a spacer, wherein the spacer is positioned between the amino acid ofthe glucagon peptide and the acyl group.

In a specific aspect of the invention, the glucagon peptide is modifiedto comprise an acyl group by direct acylation of an amine, hydroxyl, orthiol of a side chain of an amino acid of the glucagon peptide. In someembodiments, the glucagon peptide is directly acylated through the sidechain amine, hydroxyl, or thiol of an amino acid. In some embodiments,acylation is at position 10, 20, 24, 29, or 40. In this regard, theacylated glucagon peptide can comprise the amino acid sequence of SEQ IDNO: 1, or a modified amino acid sequence thereof comprising one or moreof the amino acid modifications described herein, with at least one ofthe amino acids at positions 10, 20, 24, 29, or 40 modified to any aminoacid comprising a side chain amine, hydroxyl, or thiol. In some specificembodiments of the invention, the direct acylation of the glucagonpeptide occurs through the side chain amine, hydroxyl, or thiol of theamino acid at position 10 or 40.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I:

In some exemplary embodiments, the amino acid of Formula I, is the aminoacid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II:

In some exemplary embodiments, the amino acid of Formula II is the aminoacid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III:

In some exemplary embodiments, the amino acid of Formula III is theamino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In one embodiment of the invention, the acylated glucagon peptidecomprises a spacer between the peptide and the acyl group. In someembodiments, the glucagon peptide is covalently bound to the spacer,which is covalently bound to the acyl group.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol. The aminoacid to which the spacer is attached can be any amino acid (e.g., asingly or doubly α-substituted amino acid) comprising a moiety whichpermits linkage to the spacer. For example, an amino acid comprising aside chain NH₂, —OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) issuitable. In this respect, the acylated glucagon peptide can comprisethe amino acid sequence of SEQ ID NO: 1, or a modified amino acidsequence thereof comprising one or more of the amino acid modificationsdescribed herein, with at least one of the amino acids at positions 10,20, 24, 29, and 40 modified to any amino acid comprising a side chainamine, hydroxyl, or carboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol, or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When acylation occurs through an amine group of a spacer, the acylationcan occur through the alpha amine of the amino acid or a side chainamine. In the instance in which the alpha amine is acylated, the aminoacid of the spacer can be any amino acid. For example, the amino acid ofthe spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu,Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovaleric acid,7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively, theamino acid of the spacer can be an acidic residue, e.g., Asp and Glu.

In the instance in which the side chain amine of the amino acid of thespacer is acylated, the amino acid of the spacer is an amino acidcomprising a side chain amine, e.g., an amino acid of Formula I (e.g.,Lys or Orn). In this instance, it is possible for both the alpha amineand the side chain amine of the amino acid of the spacer to be acylated,such that the glucagon peptide is diacylated. Embodiments of theinvention include such diacylated molecules.

When acylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When acylation occurs through a thiol group of a spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer.Hydrophobic bifunctional spacers are known in the art. See, e.g.,Bioconjugate Techniques, G. T. Hermanson (Academic Press, San Diego,Calif., 1996), which is incorporated by reference in its entirety. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophobic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophobic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

In some embodiments, the bifunctional spacer is not a dicarboxylic acidcomprising an unbranched, methylene of 1-7 carbon atoms between thecarboxylate groups. In some embodiments, the bifunctional spacer is adicarboxylic acid comprising an unbranched, methylene of 1-7 carbonatoms between the carboxylate groups.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms) in length. In more specific embodiments, the spacer is about 3to 10 atoms (e.g., 6 to 10 atoms) in length and the acyl group is a C12to C18 fatty acyl group, e.g., C14 fatty acyl group, C16 fatty acylgroup, such that the total length of the spacer and acyl group is 14 to28 atoms, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 atoms. In some embodiments, the length of the spacer andacyl group is 17 to 28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or naturally occurring amino acid (including,but not limited to, any of those described herein) comprising an aminoacid backbone that is 3 to 10 atoms in length (e.g., 6-amino hexanoicacid, 5-aminovaleric acid, 7-aminoheptanoic acid, and 8-aminooctanoicacid). Alternatively, the spacer can be a dipeptide or tripeptide spacerhaving a peptide backbone that is 3 to 10 atoms (e.g., 6 to 10 atoms) inlength. Each amino acid of the dipeptide or tripeptide spacer can be thesame as or different from the other amino acid(s) of the dipeptide ortripeptide and can be independently selected from the group consistingof: naturally-occurring and/or non-naturally occurring amino acids,including, for example, any of the D or L isomers of thenaturally-occurring amino acids (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr), or any D or Lisomers of the non-naturally occurring amino acids selected from thegroup consisting of: β-alanine (β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), γ-aminobutyric acid (γ-Abu), aminohexanoic acid(ε-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O₂)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid.

In some embodiments, the spacer comprises an overall negative charge,e.g., comprises one or two negatively charged amino acids. In someembodiments, the dipeptide is not any of the dipeptides of generalstructure A-B, wherein A is selected from the group consisting of Gly,Gln, Ala, Arg, Asp, Asn, Ile, Leu, Val, Phe, and Pro, wherein B isselected from the group consisting of Lys, His, Trp. In someembodiments, the dipeptide spacer is selected from the group consistingof: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyricacid-γ-aminobutyric acid, and γ-Glu-γ-Glu.

In some exemplary embodiments, the glucagon peptide is modified tocomprise an acyl group by acylation of an amine, hydroxyl, or thiol of aspacer, which spacer is attached to a side chain of an amino acid atposition 10, 20, 24, 29, or 40, or at the C-terminal amino acid of theglucagon peptide.

In yet more specific embodiments, the acyl group is attached to theamino acid at position 10 or 40 of the glucagon peptide and the lengthof the spacer and acyl group is 14 to 28 atoms. The amino acid atposition 10 or 40, in some aspects, is an amino acid of Formula I, e.g.,Lys, or a disubstituted amino acid related to Formula I. In morespecific embodiments, the glucagon peptide lacks an intramolecularbridge, e.g., a covalent intramolecular bridge. The glucagon peptide,for example, can be a peptide comprising one or more alpha,alpha-disubstituted amino acids, e.g., AIB, for stabilizing the alphahelix of the peptide. Accordingly, the acylated glucagon peptide cancomprise the amino acid sequence of any of SEQ ID NOs: 555-561 and610-612, the AIB-containing peptides of Tables 20 and 28, or of SEQ IDNOs: 657-669.

Suitable methods of peptide acylation via amines, hydroxyls, and thiolsare known in the art. See, for example, Example 19 (for methods ofacylating through an amine), Miller, Biochem Biophys Res Commun 218:377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19:54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 (1972)(for methods of acylating through a hydroxyl); and San and Silvius, JPept Res 66: 169-180 (2005) (for methods of acylating through a thiol);Bioconjugate Chem. “Chemical Modifications of Proteins: History andApplications” pages 1, 2-12 (1990); Hashimoto et al., PharmacueticalRes. “Synthesis of Palmitoyl Derivatives of Insulin and their BiologicalActivity” Vol. 6, No: 2 pp. 171-176 (1989).

The acyl group of the acylated glucagon peptide can be of any size,e.g., any length carbon chain, and can be linear or branched. In somespecific embodiments of the invention, the acyl group is a C4 to C30fatty acid. For example, the acyl group can be any of a C4 fatty acid,C6 fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fattyacid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid,C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid. Insome embodiments, the acyl group is a C8 to C20 fatty acid, e.g., a C14fatty acid or a C16 fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

In some embodiments of the invention, the glucagon peptide is modifiedto comprise an acyl group by acylation of a long chain alkane by theglucagon peptide. In specific aspects, the long chain alkane comprisesan amine, hydroxyl, or thiol group (e.g. octadecylamine, tetradecanol,and hexadecanethiol) which reacts with a carboxyl group, or activatedform thereof, of the glucagon peptide. The carboxyl group, or activatedform thereof, of the glucagon peptide can be part of a side chain of anamino acid (e.g., glutamic acid, aspartic acid) of the glucagon peptideor can be part of the peptide backbone.

In certain embodiments, the glucagon peptide is modified to comprise anacyl group by acylation of the long chain alkane by a spacer which isattached to the glucagon peptide. In specific aspects, the long chainalkane comprises an amine, hydroxyl, or thiol group which reacts with acarboxyl group, or activated form thereof, of the spacer. Suitablespacers comprising a carboxyl group, or activated form thereof, aredescribed herein and include, for example, bifunctional spacers, e.g.,amino acids, dipeptides, tripeptides, hydrophilic bifunctional spacersand hydrophobic bifunctional spacers.

As used herein, the term “activated form of a carboxyl group” refers toa carboxyl group with the general formula R(C═O)X, wherein X is aleaving group and R is the glucagon peptide or the spacer. For example,activated forms of a carboxyl groups may include, but are not limitedto, acyl chlorides, anhydrides, and esters. In some embodiments, theactivated carboxyl group is an ester with a N-hydroxysuccinimide ester(NHS) leaving group.

With regard to these aspects of the invention, in which a long chainalkane is acylated by the glucagon peptide or the spacer, the long chainalkane may be of any size and can comprise any length of carbon chain.The long chain alkane can be linear or branched. In certain aspects, thelong chain alkane is a C4 to C30 alkane. For example, the long chainalkane can be any of a C4 alkane, C6 alkane, C8 alkane, C10 alkane, C12alkane, C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22 alkane, C24alkane, C26 alkane, C28 alkane, or a C30 alkane. In some embodiments,the long chain alkane comprises a C8 to C20 alkane, e.g., a C14 alkane,C16 alkane, or a C18 alkane.

Also, in some embodiments, an amine, hydroxyl, or thiol group of theglucagon peptide is acylated with a cholesterol acid. In a specificembodiment, the glucagon peptide is linked to the cholesterol acidthrough an alkylated des-amino Cys spacer, i.e., an alkylated3-mercaptopropionic acid spacer. The alkylated des-amino Cys spacer canbe, for example, a des-amino-Cys spacer comprising a dodecaethyleneglycol moiety.

The acylated glucagon peptides described herein can be further modifiedto comprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In thisregard, the acylated glucagon peptide can comprise SEQ ID NO: 1,including any of the modifications described herein, in which at leastone of the amino acids at position 10, 20, 24, 29, and 40 comprise anacyl group and at least one of the amino acids at position 16, 17, 21,24, 29, 40, a position within a C-terminal extension, or the C-terminalamino acid are modified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and theside chain of the amino acid is covalently bonded to a hydrophilicmoiety (e.g., PEG). In some embodiments, the acyl group is attached toposition 10 or 40, optionally via a spacer comprising Cys, Lys, Orn,homo-Cys, or Ac-Phe, and the hydrophilic moiety is incorporated at a Cysresidue at position 24.

Alternatively, the acylated glucagon peptide can comprise a spacer,wherein the spacer is both acylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

In a specific aspect of the invention, the acylated glucagon peptidecomprises the amino acid sequence of any of SEQ ID NOs: 534-544, 546-549and 657-669.

Alkylation

In accordance with some embodiments, the glucagon peptide is modified tocomprise an alkyl group, e.g., an alkyl group which is notnaturally-occurring on an amino acid (e.g., an alkyl group which isnon-native to a naturally-occurring amino acid). Without being held toany particular theory, it is believed that alkylation of glucagonpeptides will achieve similar, if not the same, effects as acylation ofthe glucagon peptides, e.g., a prolonged half-life in circulation, adelayed onset of action, an extended duration of action, an improvedresistance to proteases, such as DPP-IV, and increased potency at theGLP-1 and glucagon receptors.

Alkylation can be carried out at any positions within the glucagonpeptide, including any of positions 1-29, a position within a C-terminalextension, or the C-terminal amino acid, provided that the glucagonactivity is retained. Nonlimiting examples include positions 5, 7, 10,11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24, 27, 28, 29, 30, 37, 38, 39,40, 41, 42, or 43 according to the numbering of the amino acids of SEQID NO: 1. The alkyl group can be covalently linked directly to an aminoacid of the glucagon peptide, or indirectly to an amino acid of theglucagon peptide via a spacer, wherein the spacer is positioned betweenthe amino acid of the glucagon peptide and the alkyl group. Glucagonpeptides may be alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position.Nonlimiting examples include alkylation at position 40 and pegylation atone or more positions in the C-terminal portion of the glucagon peptide,e.g., position 24, 28, 29, 40, within a C-terminal extension, or at theC-terminus (e.g., through adding a C-terminal Cys).

In a specific aspect of the invention, the glucagon peptide is modifiedto comprise an alkyl group by direct alkylation of an amine, hydroxyl,or thiol of a side chain of an amino acid of the glucagon peptide. Insome embodiments, alkylation is at position 10, 20, 24, 29, or 40. Inthis regard, the alkylated glucagon peptide can comprise the amino acidsequence of SEQ ID NO: 1, or a modified amino acid sequence thereofcomprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 10, 20, 24, 29, or 40modified to any amino acid comprising a side chain amine, hydroxyl, orthiol. In some specific embodiments of the invention, the directalkylation of the glucagon peptide occurs through the side chain amine,hydroxyl, or thiol of the amino acid at position 40.

In some embodiments, the amino acid comprising a side chain amine is anamino acid of Formula I. In some exemplary embodiments, the amino acidof Formula I, is the amino acid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula II. In some exemplary embodiments, the aminoacid of Formula II is the amino acid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula III. In some exemplary embodiments, theamino acid of Formula III is the amino acid wherein n is 1 (Cys).

In yet other embodiments, the amino acid comprising a side chain amine,hydroxyl, or thiol is a disubstituted amino acid comprising the samestructure of Formula I, Formula II, or Formula III, except that thehydrogen bonded to the alpha carbon of the amino acid of Formula I,Formula II, or Formula III is replaced with a second side chain.

In one embodiment of the invention, the alkylated glucagon peptidecomprises a spacer between the peptide and the alkyl group. In someembodiments, the glucagon peptide is covalently bound to the spacer,which is covalently bound to the alkyl group. In some exemplaryembodiments, the glucagon peptide is modified to comprise an alkyl groupby alkylation of an amine, hydroxyl, or thiol of a spacer, which spaceris attached to a side chain of an amino acid at position 10, 20, 24, 29,or 40 of the glucagon peptide. The amino acid to which the spacer isattached can be any amino acid comprising a moiety which permits linkageto the spacer. For example, an amino acid comprising a side chain NH₂,—OH, or —COOH (e.g., Lys, Orn, Ser, Asp, or Glu) is suitable. In thisrespect, the alkylated glucagon peptide can comprise the amino acidsequence of SEQ ID NO: 1, or a modified amino acid sequence thereofcomprising one or more of the amino acid modifications described herein,with at least one of the amino acids at positions 10, 20, 24, 29, or 40modified to any amino acid comprising a side chain amine, hydroxyl, orcarboxylate.

In some embodiments, the spacer is an amino acid comprising a side chainamine, hydroxyl, or thiol or a dipeptide or tripeptide comprising anamino acid comprising a side chain amine, hydroxyl, or thiol.

When alkylation occurs through an amine group of a spacer, thealkylation can occur through the alpha amine of an amino acid or a sidechain amine. In the instance in which the alpha amine is alkylated, theamino acid of the spacer can be any amino acid. For example, the aminoacid of the spacer can be a hydrophobic amino acid, e.g., Gly, Ala, Val,Leu, Ile, Trp, Met, Phe, Tyr, 6-amino hexanoic acid, 5-aminovalericacid, 7-aminoheptanoic acid, and 8-aminooctanoic acid. Alternatively,the amino acid of the spacer can be an acidic residue, e.g., Asp andGlu, provided that the alkylation occurs on the alpha amine of theacidic residue. In the instance in which the side chain amine of theamino acid of the spacer is alkylated, the amino acid of the spacer isan amino acid comprising a side chain amine, e.g., an amino acid ofFormula I (e.g., Lys or Orn). In this instance, it is possible for boththe alpha amine and the side chain amine of the amino acid of the spacerto be alkylated, such that the glucagon peptide is dialkylated.Embodiments of the invention include such dialkylated molecules.

When alkylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When alkylation occurs through a thiol group of spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In some embodiments, the spacer is a hydrophilic bifunctional spacer. Incertain embodiments, the hydrophilic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophilic bifunctional spacer is comprises a hydroxyl group and acarboxylate. In other embodiments, the hydrophilic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydrophilic bifunctional spacer comprises a thiol group and acarboxylate. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

In some embodiments, the spacer is a hydrophobic bifunctional spacer. Incertain embodiments, the hydrophobic bifunctional spacer comprises twoor more reactive groups, e.g., an amine, a hydroxyl, a thiol, and acarboxyl group or any combinations thereof. In certain embodiments, thehydrophobic bifunctional spacer comprises a hydroxyl group and acarboxylate. In other embodiments, the hydropholic bifunctional spacercomprises an amine group and a carboxylate. In other embodiments, thehydropholic bifunctional spacer comprises a thiol group and acarboxylate. Suitable hydrophobic bifunctional spacers comprising acarboxylate and a hydroxyl group or a thiol group are known in the artand include, for example, 8-hydroxyoctanoic acid and 8-mercaptooctanoicacid.

The spacer (e.g., amino acid, dipeptide, tripeptide, hydrophilicbifunctional spacer, or hydrophobic bifunctional spacer) in specificembodiments is 3 to 10 atoms (e.g., 6 to 10 atoms, (e.g., 6, 7, 8, 9, or10 atoms)) in length. In more specific embodiments, the spacer is about3 to 10 atoms (e.g., 6 to 10 atoms) in length and the alkyl is a C12 toC18 alkyl group, e.g., C14 alkyl group, C16 alkyl group, such that thetotal length of the spacer and alkyl group is 14 to 28 atoms, e.g.,about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28atoms. In some embodiments, the length of the spacer and alkyl is 17 to28 (e.g., 19 to 26, 19 to 21) atoms.

In accordance with certain foregoing embodiments, the bifunctionalspacer can be a synthetic or non-naturally occurring amino acidcomprising an amino acid backbone that is 3 to 10 atoms in length (e.g.,6-amino hexanoic acid, 5-aminovaleric acid, 7-aminoheptanoic acid, and8-aminooctanoic acid). Alternatively, the spacer can be a dipeptide ortripeptide spacer having a peptide backbone that is 3 to 10 atoms (e.g.,6 to 10 atoms) in length. The dipeptide or tripeptide spacer can becomposed of naturally-occurring and/or non-naturally occurring aminoacids, including, for example, any of the amino acids taught herein. Insome embodiments, the spacer comprises an overall negative charge, e.g.,comprises one or two negatively charged amino acids. In someembodiments, the dipeptide spacer is selected from the group consistingof: Ala-Ala, β-Ala-β-Ala, Leu-Leu, Pro-Pro, γ-aminobutyricacid-γ-aminobutyric acid, and γ-Glu-γ-Glu.

Suitable methods of peptide alkylation via amines, hydroxyls, and thiolsare known in the art. For example, a Williamson ether synthesis can beused to form an ether linkage between a hydroxyl group of the glucagonpeptide and the alkyl group. Also, a nucleophilic substitution reactionof the peptide with an alkyl halide can result in any of an ether,thioether, or amino linkage.

The alkyl group of the alkylated glucagon peptide can be of any size,e.g., any length carbon chain, and can be linear or branched. In someembodiments of the invention, the alkyl group is a C4 to C30 alkyl. Forexample, the alkyl group can be any of a C4 alkyl, C6 alkyl, C8 alkyl,C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22alkyl, C24 alkyl, C26 alkyl, C28 alkyl, or a C30 alkyl. In someembodiments, the alkyl group is a C8 to C20 alkyl, e.g., a C14 alkyl ora C16 alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

In some embodiments of the invention, the glucagon peptide is modifiedto comprise an alkyl group by reacting a nucleophilic, long chain alkanewith the glucagon peptide, wherein the glucagon peptide comprises aleaving group suitable for nucleophilic substitution. In specificaspects, the nucleophilic group of the long chain alkane comprises anamine, hydroxyl, or thiol group (e.g. octadecylamine, tetradecanol, andhexadecanethiol). The leaving group of the glucagon peptide can be partof a side chain of an amino acid or can be part of the peptide backbone.Suitable leaving groups include, for example, N-hydroxysuccinimide,halogens, and sulfonate esters.

In certain embodiments, the glucagon peptide is modified to comprise analkyl group by reacting the nucleophilic, long chain alkane with aspacer which is attached to the glucagon peptide, wherein the spacercomprises the leaving group. In specific aspects, the long chain alkanecomprises an amine, hydroxyl, or thiol group. In certain embodiments,the spacer comprising the leaving group can be any spacer discussedherein, e.g., amino acids, dipeptides, tripeptides, hydrophilicbifunctional spacers and hydrophobic bifunctional spacers furthercomprising a suitable leaving group.

With regard to these aspects of the invention, in which a long chainalkane is alkylated by the glucagon peptide or the spacer, the longchain alkane may be of any size and can comprise any length of carbonchain. The long chain alkane can be linear or branched. In certainaspects, the long chain alkane is a C4 to C30 alkane. For example, thelong chain alkane can be any of a C4 alkane, C6 alkane, C8 alkane, C10alkane, C12 alkane, C14 alkane, C16 alkane, C18 alkane, C20 alkane, C22alkane, C24 alkane, C26 alkane, C28 alkane, or a C30 alkane. In someembodiments, the long chain alkane comprises a C8 to C20 alkane, e.g., aC14 alkane, C16 alkane, or a C18 alkane.

Also, in some embodiments, alkylation can occur between the glucagonpeptide and a cholesterol moiety. For example, the hydroxyl group ofcholesterol can displace a leaving group on the long chain alkane toform a cholesterol-glucagon peptide product.

The alkylated glucagon peptides described herein can be further modifiedto comprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein. In thisregard, the alkylated glucagon peptide can comprise SEQ ID NO: 1 or amodified amino acid sequence thereof comprising one or more of the aminoacid modifications described herein, in which at least one of the aminoacids at position 10, 20, 24, 29, or 40 comprise an alkyl group and atleast one of the amino acids at position 16, 17, 21, 24, 29, 40, aposition within a C-terminal extension or the C-terminal amino acid aremodified to a Cys, Lys, Orn, homo-Cys, or Ac-Phe, and the side chain ofthe amino acid is covalently bonded to a hydrophilic moiety (e.g., PEG).In some embodiments, the alkyl group is attached to position 40,optionally via a spacer comprising Cys, Lys, Orn, homo-Cys, or Ac-Phe,and the hydrophilic moiety is incorporated at a Cys residue at position24.

Alternatively, the alkylated glucagon peptide can comprise a spacer,wherein the spacer is both alkylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

C-Terminal Truncation

In some embodiments, the glucagon peptides described herein are furthermodified by truncation or deletion of one or two amino acids of theC-terminus of the glucagon peptide (i.e., position 29 and/or 28) withoutaffecting activity and/or potency at the glucagon and GLP-1 receptors.In this regard, the glucagon peptide can comprise amino acids 1-27 or1-28 of the native glucagon peptide (SEQ ID NO: 1), optionally with oneor more modifications described herein.

In one embodiment, the truncated glucagon agonist peptide comprises SEQID NO: 550 or SEQ ID NO: 551. In another embodiment, the truncatedglucagon agonist peptide comprises SEQ ID NO: 552 or SEQ ID NO: 553.

Charged C-Terminal Residues

The solubility of the glucagon peptide of SEQ ID NO: 20 can be furtherimproved, for example, by introducing one, two, three or more chargedamino acid(s) to the C-terminal portion of glucagon peptide of SEQ IDNO: 20, preferably at a position C-terminal to position 27. Such acharged amino acid can be introduced by substituting a native amino acidwith a charged amino acid, e.g. at positions 28 or 29, or alternativelyby adding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. Alternatively, solubility can also be enhancedby covalently linking hydrophilic moieties, such as polyethylene glycol,to the peptide.

Exemplary Embodiments

In accordance with one embodiment, a glucagon analog is providedcomprising the sequence of SEQ ID NO: 55, wherein said analog differsfrom SEQ ID NO: 55 by 1 to 3 amino acids, selected from positions 1, 2,3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 24, 27, 28, and 29, whereinsaid glucagon peptide exhibits at least 20% of the activity of nativeGLP-1 at the GLP-1 receptor.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising the sequence:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 33) wherein the Xaa at position 15 is selected from thegroup of amino acids consisting of Asp, Glu, cysteic acid, homoglutamicacid and homocysteic acid, Xaa at position 16 is selected from the groupof amino acids consisting of Ser, Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaa at position 20 is Gln or Lys, the Xaa atposition 24 is Gln or Glu, the Xaa at position 28 is Asn, Lys or anacidic amino acid, the Xaa at position 29 is Thr, Gly or an acidic aminoacid, and R is COOH or CONH₂, with the proviso that when position 16 isserine, position 20 is Lys, or alternatively when position 16 is serinethe position 24 is Glu and either position 20 or position 28 is Lys. Inone embodiment the glucagon/GLP-1 receptor co-agonist comprises thesequence of SEQ ID NO: 33 wherein the amino acid at position 28 isaspartic acid and the amino acid at position 29 is glutamic acid. Inanother embodiment the amino acid at position 28 is the nativeasparagine, the amino acid at position 29 is glycine and the amino acidsequence of SEQ ID NO: 29 or SEQ ID NO: 65 is covalently linked to thecarboxy terminus of SEQ ID NO: 33.

In one embodiment a co-agonist is provided comprising the sequence ofSEQ ID NO: 33 wherein an additional acidic amino acid added to thecarboxy terminus of the peptide. In a further embodiment the carboxyterminal amino acid of the glucagon analog has an amide in place of thecarboxylic acid group of the natural amino acid. In one embodiment theglucagon analog comprises a sequence selected from the group consistingof SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ IDNO: 44.

In accordance with one embodiment a glucagon peptide analog of SEQ IDNO: 33 is provided, wherein said analog differs from SEQ ID NO: 33 by 1to 3 amino acids, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14,17, 18, 19, 21 and 27, with the proviso that when the amino acid atposition 16 is serine, either position 20 is lysine, or a lactam bridgeis formed between the amino acid at position 24 and either the aminoacid at position 20 or position 28. In accordance with one embodimentthe analog differs from SEQ ID NO: 33 by 1 to 3 amino acids selectedfrom positions 1, 2, 3, 21 and 27. In one embodiment the glucagonpeptide analog of SEQ ID NO: 33 differs from that sequence by 1 to 2amino acids, or in one embodiment by a single amino acid, selected formpositions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, with theproviso that when the amino acid at position 16 is serine, eitherposition 20 is lysine, or a lactam bridge is formed between the aminoacid at position 24 and either the amino acid at position 20 or position28.

In accordance with another embodiment a relatively selective GLP-1receptor agonist is provided comprising the sequenceNH2-His-Ser-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 53) wherein the Xaa at position 3 is selected from the groupof amino acids consisting of Glu, Orn or Nle, the Xaa at position 15 isselected from the group of amino acids consisting of Asp, Glu, cysteicacid, homoglutamic acid and homocysteic acid, Xaa at position 16 isselected from the group of amino acids consisting of Ser, Glu, Gln,homoglutamic acid and homocysteic acid, the Xaa at position 20 is Gln orLys, the Xaa at position 24 is Gln or Glu, the Xaa at position 28 isAsn, Lys or an acidic amino acid, the Xaa at position 29 is Thr, Gly oran acidic amino acid, and R is COOH, CONH₂, SEQ ID NO: 26 or SEQ ID NO:29, with the proviso that when position 16 is serine, position 20 isLys, or alternatively when position 16 is serine the position 24 is Gluand either position 20 or position 28 is Lys. In one embodiment theamino acid at position 3 is glutamic acid. In one embodiment the acidicamino acid substituted at position 28 and/or 29 is aspartic acid orglutamic acid. In one embodiment the glucagon peptide, including aco-agonist peptide, comprises the sequence of SEQ ID NO: 33 furthercomprising an additional acidic amino acid added to the carboxy terminusof the peptide. In a further embodiment the carboxy terminal amino acidof the glucagon analog has an amide in place of the carboxylic acidgroup of the natural amino acid.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising a modified glucagon peptide selected from thegroup consisting of:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 34), wherein the Xaa at position 15 is selected from thegroup of amino acids consisting of Asp, Glu, cysteic acid, homoglutamicacid and homocysteic acid, Xaa at position 16 is selected from the groupof amino acids consisting of Ser, Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaa at position 20 is Gln or Lys, the Xaa atposition 24 is Gln or Glu and the Xaa at position 28 is Asn, Asp or Lys,R is COOH or CONH₂, the Xaa at position 29 is Thr or Gly, and R is COOH,CONH₂, SEQ ID NO: 26 or SEQ ID NO: 29, with the proviso that whenposition 16 is serine, position 20 is Lys, or alternatively whenposition 16 is serine the position 24 is Glu and either position 20 orposition 28 is Lys. In one embodiment R is CONH₂, the Xaa at position 15is Asp, the Xaa at position 16 is selected from the group of amino acidsconsisting of Glu, Gln, homoglutamic acid and homocysteic acid, the Xaasat positions 20 and 24 are each Gln the Xaa at position 28 is Asn or Aspand the Xaa at position 29 is Thr. In one embodiment the Xaas atpositions 15 and 16 are each Glu, the Xaas at positions 20 and 24 areeach Gln, the Xaa at position 28 is Asn or Asp, the Xaa at position 29is Thr and R is CONH₂.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, applicants anticipate that one or moreof the amino acids located at positions at positions 2, 5, 7, 10, 11,12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide of SEQID NO: 11 can be substituted with an amino acid different from thatpresent in the native glucagon peptide, and still retain activity at theglucagon receptor. In one embodiment the methionine residue present atposition 27 of the native peptide is changed to leucine or norleucine toprevent oxidative degradation of the peptide. In another embodiment theamino acid at position 20 is substituted with Lys, Arg, Orn orCitrullene and/or position 21 is substituted with Glu, homoglutamic acidor homocysteic acid.

In one embodiment a glucagon analog of SEQ ID NO: 20 is provided wherein1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11, 13, 14,17, 18, 19, 21, 27, 28 or 29 of the analog differ from the correspondingamino acid of SEQ ID NO: 1, with the proviso that when the amino acid atposition 16 is serine, position 20 is Lys, or alternatively whenposition 16 is serine the position 24 is Glu and either position 20 orposition 28 is Lys. In accordance with another embodiment a glucagonanalog of SEQ ID NO: 20 is provided wherein 1 to 3 amino acids selectedfrom positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27, 28 or29 of the analog differ from the corresponding amino acid of SEQ IDNO: 1. In another embodiment, a glucagon analog of SEQ ID NO: 8, SEQ IDNO: 9 or SEQ ID NO: 11 is provided wherein 1 to 2 amino acids selectedfrom positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20 or 21 of theanalog differ from the corresponding amino acid of SEQ ID NO: 1, and ina further embodiment the one to two differing amino acids representconservative amino acid substitutions relative to the amino acid presentin the native glucagon sequence (SEQ ID NO: 1). In one embodiment aglucagon peptide of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQID NO: 15 is provided wherein the glucagon peptide further comprisesone, two or three amino acid substitutions at positions selected frompositions 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27 or 29. In oneembodiment the substitutions at positions 2, 5, 7, 10, 11, 13, 14, 16,17, 18, 19, 20, 21, 27 or 29 are conservative amino acid substitutions.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising a variant of the sequence of SEQ ID NO 33,wherein 1 to 10 amino acids selected from positions 16, 17, 18, 20, 21,23, 24, 27, 28 and 29, respectively, of the variant differ from thecorresponding amino acid of SEQ ID NO: 1. In accordance with oneembodiment a variant of the sequence of SEQ ID NO 33 is provided whereinthe variant differs from SEQ ID NO: 33 by one or more amino acidsubstitutions selected from the group consisting of Gln17, Ala18, Glu21,Ile23, Ala24, Val27 and Gly29. In accordance with one embodiment aglucagon/GLP-1 receptor co-agonist is provided comprising variants ofthe sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected frompositions 17-26 of the variant differ from the corresponding amino acidof SEQ ID NO: 1. In accordance with one embodiment a variant of thesequence of SEQ ID NO 33 is provided wherein the variant differs fromSEQ ID NO: 33 by an amino acid substitution selected from the groupconsisting of Gln17, Ala18, Glu21, Ile23 and Ala24. In accordance withone embodiment a variant of the sequence of SEQ ID NO 33 is providedwherein the variant differs from SEQ ID NO: 33 by an amino acidsubstitution at position 18 wherein the substituted amino acid isselected from the group consisting of Ala, Ser, Thr, and Gly. Inaccordance with one embodiment a variant of the sequence of SEQ ID NO 33is provided wherein the variant differs from SEQ ID NO: 33 by an aminoacid substitution of Ala at position 18. Such variations are encompassedby SEQ ID NO: 55. In another embodiment a glucagon/GLP-1 receptorco-agonist is provided comprising variants of the sequence of SEQ ID NO33, wherein 1 to 2 amino acids selected from positions 17-22 of thevariant differ from the corresponding amino acid of SEQ ID NO: 1, and ina further embodiment a variant of SEQ ID NO 33 is provided wherein thevariant differs from SEQ ID NO: 33 by for 2 amino acid substitutions atpositions 20 and 21. In accordance with one embodiment a glucagon/GLP-1receptor co-agonist is provided comprising the sequence:NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Xaa-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 51), wherein the

Xaa at position 15 is Asp, Glu, cysteic acid, homoglutamic acid orhomocysteic acid, the Xaa at position 16 is Ser, Glu, Gln, homoglutamicacid or homocysteic acid, the Xaa at position 20 is Gln, Lys, Arg, Ornor citrulline, the Xaa at position 21 is Asp, Glu, homoglutamic acid orhomocysteic acid, the Xaa at position 24 is Gln or Glu, the Xaa atposition 28 is Asn, Lys or an acidic amino acid, the Xaa at position 29is Thr or an acid amino acid and R is COOH or CONH₂. In one embodiment Ris CONH₂. In accordance with one embodiment a glucagon/GLP-1 receptorco-agonist is provided comprising a variant of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 47, SEQ IDNO: 48 or SEQ ID NO: 49, wherein the variant differs from said sequenceby an amino acid substitution at position 20. In one embodiment theamino acid substitution is selected form the group consisting of Lys,Arg, Orn or citrulline for position 20.

In one embodiment a glucagon agonist is provided comprising an analogpeptide of SEQ ID NO: 34 wherein the analog differs from SEQ ID NO: 34by having an amino acid other than serine at position 2. In oneembodiment the serine residue is substituted with aminoisobutyric acid,D-alanine, and in one embodiment the serine residue is substituted withaminoisobutyric acid. Such modifications suppresses cleavage bydipeptidyl peptidase IV while retaining the inherent potency of theparent compound (e.g. at least 75, 80, 85, 90, 95% or more of thepotency of the parent compound). In one embodiment the solubility of theanalog is increased, for example, by introducing one, two, three or morecharged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. In exemplaryembodiments, one, two, three or all of the charged amino acids arenegatively charged. In another embodiment the analog further comprisesan acidic amino acid substituted for the native amino acid at position28 or 29 or an acidic amino acid added to the carboxy terminus of thepeptide of SEQ ID NO: 34.

In one embodiment the glucagon analogs disclosed herein are furthermodified at position 1 or 2 to reduce susceptibility to cleavage bydipeptidyl peptidase IV. In one embodiment a glucagon analog of SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQID NO: 15 is provided wherein the analog differs from the parentmolecule by a substitution at position 2 and exhibits reducedsusceptibility (i.e., resistance) to cleavage by dipeptidyl peptidaseIV. More particularly, in one embodiment position 2 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of D-serine, D-alanine, valine, amino n-butyric acid,glycine, N-methyl serine and aminoisobutyric acid. In one embodimentposition 2 of the analog peptide is substituted with an amino acidselected from the group consisting of D-serine, D-alanine, glycine,N-methyl serine and aminoisobutyric acid. In another embodiment position2 of the analog peptide is substituted with an amino acid selected fromthe group consisting of D-serine, glycine, N-methyl serine andaminoisobutyric acid. In one embodiment the glucagon peptide comprisesthe sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

In one embodiment a glucagon analog of SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is providedwherein the analog differs from the parent molecule by a substitution atposition 1 and exhibits reduced susceptibility (i.e., resistance) tocleavage by dipeptidyl peptidase IV. More particularly, position 1 ofthe analog peptide is substituted with an amino acid selected from thegroup consisting of D-histidine, alpha,alpha-dimethyl imidiazole aceticacid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazoleacetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. In another embodiment a glucagon agonist is providedcomprising an analog peptide of SEQ ID NO: 34 wherein the analog differsfrom SEQ ID NO: 34 by having an amino acid other than histidine atposition 1. In one embodiment the solubility of the analog is increased,for example, by introducing one, two, three or more charged aminoacid(s) to the C-terminal portion of native glucagon, preferably at aposition C-terminal to position 27. In exemplary embodiments, one, two,three or all of the charged amino acids are negatively charged. Inanother embodiment the analog further comprises an acidic amino acidsubstituted for the native amino acid at position 28 or 29 or an acidicamino acid added to the carboxy terminus of the peptide of SEQ ID NO:34. In one embodiment the acidic amino acid is aspartic acid or glutamicacid.

In one embodiment the glucagon/GLP-1 receptor co-agonist comprises asequence of SEQ ID NO: 20 further comprising an additional carboxyterminal extension of one amino acid or a peptide selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. Inthe embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in one embodiment the additionalcarboxy terminus amino acid has an amide group in place of thecarboxylic acid of the native amino acid. In one embodiment theadditional amino acid is selected from the group consisting of glutamicacid, aspartic acid and glycine.

In an alternative embodiment a glucagon/GLP-1 receptor co-agonist isprovided wherein the peptide comprises at least one lactam ring formedbetween the side chain of a glutamic acid residue and a lysine residue,wherein the glutamic acid residue and a lysine residue are separated bythree amino acids. In one embodiment the carboxy terminal amino acid ofthe lactam bearing glucagon peptide has an amide group in place of thecarboxylic acid of the native amino acid. More particularly, in oneembodiment a glucagon and GLP-1 co-agonist is provided comprising amodified glucagon peptide selected from the group consisting of:

(SEQ ID NO: 66)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 67)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 68)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 69)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Xaa-R (SEQ ID NO: 16)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Asn-Thr-R (SEQ ID NO: 17)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R (SEQ ID NO: 18)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-Rwherein Xaa at position 28=Asp, or Asn, the Xaa at position 29 is Thr orGly, R is selected from the group consisting of COOH, CONH₂, glutamicacid, aspartic acid, glycine, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ IDNO: 28, and a lactam bridge is formed between Lys at position 12 and Gluat position 16 for SEQ ID NO: 66, between Glu at position 16 and Lys atposition 20 for SEQ ID NO: 67, between Lys at position 20 and Glu atposition 24 for SEQ ID NO: 68, between Glu at position 24 and Lys atposition 28 for SEQ ID NO: 69, between Lys at position 12 and Glu atposition 16 and between Lys at position 20 and Glu at position 24 forSEQ ID NO: 16, between Lys at position 12 and Glu at position 16 andbetween Glu at position 24 and Lys at position 28 for SEQ ID NO: 17 andbetween Glu at position 16 and Lys at position 20 and between Glu atposition 24 and Lys at position 28 for SEQ ID NO: 18. In one embodimentR is selected from the group consisting of COOH, CONH₂, glutamic acid,aspartic acid, glycine, the amino acid at position 28 is Asn, and theamino acid at position 29 is threonine. In one embodiment R is CONH₂,the amino acid at position 28 is Asn and the amino acid at position 29is threonine. In another embodiment R is selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 65 and theamino acid at position 29 is glycine.

In a further embodiment the glucagon/GLP-1 receptor co-agonist isselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17and SEQ ID NO: 18, wherein the peptide further comprises an additionalcarboxy terminal extension of one amino acid or a peptide selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.In one embodiment the terminal extension comprises the sequence of SEQID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 and the glucagon peptidecomprises the sequence of SEQ ID NO: 55. In one embodiment theglucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO:33 wherein the amino acid at position 16 is glutamic acid, the aminoacid at position 20 is lysine, the amino acid at position 28 isasparagine and the amino acid sequence of SEQ ID No: 26 or SEQ ID NO: 29is linked to the carboxy terminus of SEQ ID NO: 33.

In the embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in one embodiment the amino acid hasan amide group in place of the carboxylic acid of the native amino acid.In one embodiment the additional amino acid is selected from the groupconsisting of glutamic acid and aspartic acid and glycine. In theembodiments wherein the glucagon agonist analog further comprises acarboxy terminal extension, the carboxy terminal amino acid of theextension, in one embodiment, ends in an amide group or an ester grouprather than a carboxylic acid.

In another embodiment the glucagon/GLP-1 receptor co-agonist comprisesthe sequence:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Xaa-CONH₂(SEQ ID NO: 19), wherein the Xaa at position 30 represents any aminoacid. In one embodiment Xaa is selected from one of the 20 common aminoacids, and in one embodiment the amino acid is glutamic acid, asparticacid or glycine. The solubility of this peptide can be further improvedby covalently linking a PEG chain to the side chain of amino acid atposition 17, 21, 24 or 30 of SEQ ID NO: 19. In a further embodiment thepeptide comprises an additional carboxy terminal extension of a peptideselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28. In accordance with one embodiment the glucagon/GLP-1receptor co-agonist comprises the sequence of SEQ ID NO: 30, SEQ ID NO:31 and SEQ ID NO: 32.

Additional site specific modifications internal to the glucagon sequenceof SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 andSEQ ID NO: 64 can be made to yield a set of glucagon agonists thatpossess variable degrees of GLP-1 agonism. Accordingly, peptides thatpossess virtually identical in vitro potency at each receptor have beenprepared and characterized. Similarly, peptides with tenfold selectivelyenhanced potency at each of the two receptors have been identified andcharacterized. As noted above substitution of the serine residue atposition 16 with glutamic acid enhances the potency of native glucagonat both the Glucagon and GLP-1 receptors, but maintains approximately atenfold selectivity for the glucagon receptor. In addition bysubstituting the native glutamine at position 3 with glutamic acid (SEQID NO: 22) generates a glucagon analog that exhibits approximately atenfold selectivity for the GLP-1 receptor.

The solubility of the glucagon/GLP-1 co-agonist peptides can be furtherenhanced in aqueous solutions at physiological pH, while retaining thehigh biological activity relative to native glucagon by the introductionof hydrophilic groups at positions 16, 17, 21, and 24 of the peptide, orby the addition of a single modified amino acid (i.e., an amino acidmodified to comprise a hydrophilic group) at the carboxy terminus of theglucagon/GLP-1 co-agonist peptide. In accordance with one embodiment thehydrophilic group comprises a polyethylene (PEG) chain. Moreparticularly, in one embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18 wherein a PEG chain is covalently linked to the side chain of anamino acids at position 16, 17, 21, 24, 29 or the C-terminal amino acidof the glucagon peptide, with the proviso that when the peptidecomprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13the polyethylene glycol chain is covalently bound to an amino acidresidue at position 17, 21 or 24, when the peptide comprises SEQ ID NO:14 or SEQ ID NO: 15 the polyethylene glycol chain is covalently bound toan amino acid residue at position 16, 17 or 21, and when the peptidecomprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 the polyethyleneglycol chain is covalently bound to an amino acid residue at position 17or 21.

In one embodiment the glucagon peptide comprises the sequence of SEQ IDNO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 17,21, 24, or the C-terminal amino acid of the glucagon peptide, and thecarboxy terminal amino acid of the peptide has an amide group in placeof the carboxylic acid group of the native amino acid. In one embodimentthe glucagon/GLP-1 receptor co-agonist peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18and SEQ ID NO: 19, wherein a PEG chain is covalently linked to the sidechain of an amino acid at position 17, 21 or 24 of SEQ ID NO: 12, SEQ IDNO: 13 and SEQ ID NO: 19, or at position 16, 17 or 21 of SEQ ID NO: 14and SEQ ID NO: 15 or at position 17 or 21 of SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18 of the glucagon peptide. In another embodiment theglucagon/GLP-1 receptor co-agonist peptide comprises the sequence of SEQID NO: 11 or SEQ ID NO: 19, wherein a PEG chain is covalently linked tothe side chain of an amino acids at position 17, 21 or 24 or theC-terminal amino acid of the glucagon peptide.

In accordance with one embodiment, and subject to the provisolimitations described in the preceding paragraphs, the glucagonco-agonist peptide is modified to contain one or more amino acidsubstitution at positions 16, 17, 21, 24, or 29 or the C-terminal aminoacid, wherein the native amino acid is substituted with an amino acidhaving a side chain suitable for crosslinking with hydrophilic moieties,including for example, PEG. The native peptide can be substituted with anaturally occurring amino acid or a synthetic (non-naturally occurring)amino acid. Synthetic or non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.Alternatively, the amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG, canbe added to the carboxy terminus of any of the glucagon analogsdisclosed herein. In accordance with one embodiment an amino acidsubstitution is made in the glucagon/GLP-1 receptor co-agonist peptideat a position selected from the group consisting of 16, 17, 21, 24, or29 replacing the native amino acid with an amino acid selected from thegroup consisting of lysine, cysteine, ornithine, homocysteine and acetylphenylalanine, wherein the substituting amino acid further comprises aPEG chain covalently bound to the side chain of the amino acid. In oneembodiment a glucagon peptide selected form the group consisting of SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 is further modified tocomprise a PEG chain is covalently linked to the side chain of an aminoacid at position 17 or 21 of the glucagon peptide. In one embodiment thepegylated glucagon/GLP-1 receptor co-agonist further comprises thesequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55 or SEQ ID NO: 56, further comprising a C-terminal extension ofSEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 linked to the C-terminalamino acid of SEQ ID NO: 55 or SEQ ID NO: 56, and optionally furthercomprising a PEG chain covalently linked to the side chain of an aminoacids at position 17, 18, 21, 24 or 29 or the C-terminal amino acid ofthe peptide. In another embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 55 or SEQ ID NO: 56, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 21 or24 of the glucagon peptide and the peptide further comprises aC-terminal extension of SEQ ID NO: 26, or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55, or SEQ ID NO: 33 or SEQ ID NO: 34, wherein an additionalamino acid is added to the carboxy terminus of SEQ ID NO: 33 or SEQ IDNO: 34, and a PEG chain is covalently linked to the side chain of theadded amino acid. In a further embodiment, the pegylated glucagon analogfurther comprises a C-terminal extension of SEQ ID NO: 26 or SEQ ID NO:29 linked to the C-terminal amino acid of SEQ ID NO: 33 or SEQ ID NO:34. In another embodiment the glucagon peptide comprises the sequence ofSEQ ID NO: 19, wherein a PEG chain is covalently linked to the sidechain of the amino acid at position 30 of the glucagon peptide and thepeptide further comprises a C-terminal extension of SEQ ID NO: 26 or SEQID NO: 29 linked to the C-terminal amino acid of SEQ ID NO: 19.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 10,000 Daltons. In one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 1,000 to about 5,000 Daltons. In an alternative embodiment thepolyethylene glycol chain has an average molecular weight selected fromthe range of about 10,000 to about 20,000 Daltons. In accordance withone embodiment the pegylated glucagon peptide comprises two or morepolyethylene chains covalently bound to the glucagon peptide wherein thetotal molecular weight of the glucagon chains is about 1,000 to about5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide consisting of SEQ ID NO: 5 or a glucagon agonistanalog of SEQ ID NO: 5, wherein a PEG chain is covalently linked to theamino acid residue at position 21 and at position 24, and wherein thecombined molecular weight of the two PEG chains is about 1,000 to about5,000 Daltons.

In certain exemplary embodiments, the glucagon peptide comprises theamino acid sequence of SEQ ID NO: 1 with up to ten amino acidmodifications and comprises an amino acid at position 10 which isacylated or alkylated. In some embodiments, the amino acid at position10 is acylated or alkylated with a C4 to C30 fatty acid. In certainaspects, the amino acid at position 10 comprises an acyl group or analkyl group which is non-native to a naturally-occurring amino acid.

In certain embodiments, the glucagon peptide comprising an amino acid atposition 10 which is acylated or alkylated comprises a stabilized alphahelix. Accordingly, in certain aspects, the glucagon peptide comprisesan acyl or alkyl group as described herein and an intramolecular bridge,e.g., a covalent intramolecular bridge (e.g., a lactam bridge) betweenthe side chains of an amino acid at position i and an amino acid atposition i+4, wherein i is 12, 16, 20, or 24. Alternatively oradditionally, the glucagon peptide comprises an acyl or alkyl group asdescribed herein and one, two, three or more of positions 16, 20, 21and/or 24 of the glucagon peptide are substituted with anα,α-disubstituted amino acid, e.g., AIB. In some instances, thenon-native glucagon peptide comprises Glu at position 16 and Lys atposition 20, wherein optionally a lactam bridge lnkes the Glu and theLys, and, optionally, the glucagon peptide further comprises one or moremodifications selected from the group consisting of: Gln at position 17,Ala at position 18, Glu at position 21, Ile at position 23, and Ala atposition 24.

Also, in any of the embodiments, wherein the glucagon peptide comprisesan amino acid at position 10 which is acylated or alkylated, theglucagon peptide can further comprise a C-terminal amide in lieu of theC-terminal alpha carboxylate.

In some embodiments, the glucagon peptide comprising an acyl or alkylgroup as described herein further comprises an amino acid substitutionat position 1, at position 2, or at positions 1 and 2, wherein the aminoacid substitution(s) achieve DPP-IV protease resistance. For example,the His at position 1 may be substituted with an amino acid selectedfrom the group consisting of: D-histidine, alpha,alpha-dimethylimidiazole acetic acid (DMIA), N-methyl histidine, alpha-methylhistidine, imidazole acetic acid, desaminohistidine, hydroxyl-histidine,acetyl-histidine and homo-histidine. Alternatively or additionally, theSer at position 2 may be substituted with an amino acid selected fromthe group consisting of: D-serine, alanine, D-alanine, valine, glycine,N-methyl serine, N-methyl alanine, and amino isobutyric acid.

The glucagon peptide comprising the amino acid at position 10 which isacylated or alkylated as described herein can comprise any amino acidsequence which is substantially related to SEQ ID NO: 1. For instance,the glucagon peptide comprises SEQ ID NO: 1 with up to 10 amino acidmodifications (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 modifications). Incertain embodiments, the amino acid sequence of the acylated oralkylated glucagon peptide is greater than 25% identical to SEQ ID NO: 1(e.g., greater than 30%, 35%, 40%, 50%, 60%, 70% 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or nearly 100% identical to SEQ ID NO: 1). Incertain specific embodiments, the glucagon peptide is one whichcomprises SEQ ID NOs: 55 with an amino acid at position 10 acylated oralkylated as described herein. The glucagon peptide can be any of SEQ IDNOs: 55, 55 with 1 or 2 amino acid modifications, 2-4, 9-18, 20, 23-25,33, 40-44, 53, 56, 61, 62, 64, 66-514, and 534.

The acyl or alkyl group of these embodiments may be any acyl or alkylgroup described herein. For example, the acyl group may be a C4 to C30(e.g., C8 to C24) fatty acyl group and the alkyl group may be a C4 toC30 (e.g., C8 to C24) alkyl group.

The amino acid to which the acyl or alkyl group is attached may be anyof the amino acids described herein, e.g., an amino acid of any ofFormula I (e.g., Lys), Formula II, and Formula III.

In some embodiments, the acyl group or alkyl group is directly attachedto the amino acid at position 10. In some embodiments, the acyl or alkylgroup is attached to the amino acid at position 10 via a spacer, suchas, for example, a spacer which is 3 to 10 atoms in length, e.g., anamino acid or dipeptide. Suitable spacers for purposes of attaching anacyl or alkyl group are described herein.

In some embodiments of the invention, an analog of a glucagon peptide,which analog exhibits agonist activity at the GIP receptor, is provided.The analog in certain embodiments comprises the amino acid sequence ofSEQ ID NO: 1 with at least one amino acid modification (optionally, upto 15 amino acid modifications), and an extension of 1 to 21 amino acidsC-terminal to the amino acid at position 29 of the analog.

In certain aspects, the analogs comprise at least one amino acidmodification and up to 15 amino acid modifications (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, amino acid modifications, up to 10 aminoacid modifications). In certain embodiments, the analogs comprise atleast one amino acid modification at up to 10 amino acid modificationsand additional conservative amino acid modifications. Conservative aminoacid modifications are described herein.

In some aspects, at least one of the amino acid modifications confers astabilized alpha helix structure in the C-terminal portion of theanalog. Modifications which achieve a stabilized alpha helix structureare described herein. See, for example, the teachings under the sectionentitled Stabilization of the alpha helix/Intramolecular bridges. Insome aspects, the analog comprises an intramolecular bridge (e.g., acovalent intramolecular bridge, a non-covalent intramolecular bridge)between the side chains of two amino acids of the analog. In certainaspects, an intramolecular bridge links the side chains of the aminoacids at positions i and i+4, wherein i is 12, 13, 16, 17, 20, or 24. Inother aspects, an intramolecular bridge connects the side chains of theamino acids at positions j and j+3, wherein j is 17, or at positions kand k+7″ wherein k is any integer between 12 and 22. In certainembodiments, the intramolecular bridge is a covalent intramolecularbridge, e.g., a lactam bridge. In specific aspects, the lactam bridgeconnects the side chains of the amino acids at positions 16 and 20. Inparticular aspects, one of the amino acids at positions 16 and 20 is apositive-charged amino acid and the other is a negative-charged aminoacid. For example, the analog can comprise a lactam bridge connectingthe side chains of a Glu at position 16 and a Lys at position 20. Inother aspects, the negative-charged amino acid and the positive-chargedamino acid form a salt bridge. In this instance, the intramolecularbridge is a non-covalent intramolecular bridge.

In particular aspects, the amino acid modification which confers astabilized alpha helix is an insertion or substitution of an amino acidof SEQ ID NO: 1 with an α,α-disubstituted amino acid. Suitableα,α-disubstituted amino acids for purposes of stabilizing the alphahelix are described herein and include, for example, AIB. In someaspects, one, two, three, or more of the amino acids at positions 16,20, 21, and 24 of SEQ ID NO: 1 are substituted with an α,α-disubstitutedamino acid, e.g., AIB. In particular embodiments, the amino acid atposition 16 is AIB.

The analog which exhibits agonist activity at the GIP receptor cancomprise additional modifications, such as any of those describedherein. For instance, the amino acid modifications may increase ordecrease activity at one or both of the GLP-1 receptor and glucagonreceptor. The amino acid modifications may increase stability of thepeptide, e.g., increase resistance to DPP-IV protease degradation,stabilize the bond between amino acids 15 and 16. The amino acidmodifications may increase the solubility of the peptide and/or alterthe time of action of the analog at any of the GIP, glucagon, and GLP-1receptors. A combination of any of these types of modifications may bepresent in the analogs which exhibit agonist activity at the GIPreceptor.

Accordingly, in some aspects, the analog comprises the amino acidsequence of SEQ ID NO: 1 with one or more of: Gln at position 17, Ala atposition 18, Glu at position 21, Ile at position 23, and Ala, Asn, orCys at position 24, or conservative amino acid substitutions thereof. Insome aspects, the analog comprises a C-terminal amide in place of theC-terminal alpha carboxylate. In certain embodiments, the analogcomprises an amino acid substitution at position 1, position 2, orpositions 1 and 2, which substitution(s) achieve DPP-IV proteaseresistance. Suitable amino acid substitutions are described herein. Forexample, DMIA at position 1 and/or d-Ser or AIB at position 2.

In some embodiments, the analog is modified at positions 27 and/or 28,and optionally at position 29. For example, the Met at position 27 issubstituted with a large aliphatic amino acid, optionally Leu, the Asnat position 28 is substituted with a small aliphatic amino acid,optionally Ala, and the Thr at position 29 is substituted with a smallaliphatic amino acid, optionally Gly. Without being bound to anyparticular theory, it is believed that substitution with LAG atpositions 27-29 provides increased GIP activity relative to the nativeMNT sequence of SEQ ID NO: 1 at those positions. In some aspects, theamino acid at position 1 is an amino acid comprising an imidazole ring,e.g., His, analogs of His, and the analog is modified at positions 27and/or 28, and optionally at position 29, as described herein.

Additionally or alternatively, the analog may comprise one or acombination of: (a) Ser at position 2 substituted with Ala; (b) Gln atposition 3 substituted with Glu or a glutamine analog; (c) Thr atposition 7 substituted with a Ile; (d) Tyr at position 10 substitutedwith Trp or an amino acid comprising an acyl or alkyl group which isnon-native to a naturally-occurring amino acid; (e) Lys at position 12substituted with Ile; (f) Asp at position 15 substituted with Glu; (g)Ser at position 16 substituted with Glu; (h) Gln at position 20substituted with Ser, Thr, Ala, AIB; (i) Gln at position 24 substitutedwith Ser, Thr, Ala, AIB; (j) Met at position 27 substituted with Leu orNle; (k) Asn at position 29 substituted with a charged amino acid,optionally, Asp or Glu; and (l) Thr at position 29 substituted with Glyor a charged amino acid, optionally, Asp or Glu.

In certain aspects, the analog does not comprise an amino acidmodification at position 1 which modification confers GIP agonistactivity. In some aspects, the amino acid at position 1 is not a large,aromatic amino acid, e.g., Tyr. In some embodiments, the amino acid atposition 1 is an amino acid comprising an imidazole ring, e.g., His,analogs of His. In certain embodiments, the analog is not any of thecompounds disclosed in U.S. Patent Application No. 61/151,349. Incertain aspects, the analog comprises the amino acid sequence of any ofSEQ ID NOs: 657-669. In certain aspects, the analog comprises a modifiedamino acid sequence of any of SEQ ID NOs: 657-669 in which the aminoacid at position 12 is Ile and/or the amino acid at position 27 is Leuand/or the amino acid at position 28 is Ala. In some aspects, the analogcomprises the amino acid sequence of any of SEQ ID NOs: 676, 677, 679,680

With regard to the analogs which exhibit agonist activity at the GIPreceptor, the analog comprises an extension of 1-21 amino acids (e.g.,5-19, 7-15, 9-12 amino acids). The extension of the analog may compriseany amino acid sequence, provided that the extension is 1 to 21 aminoacids. In some aspects, the extension is 7 to 15 amino acids and inother aspects, the extension is 9 to 12 amino acids. In someembodiments, the extension comprises (i) the amino acid sequence of SEQID NO: 26 or 674, (ii) an amino acid sequence which has high sequenceidentity (e.g., at least 80%, 85%, 90%, 95%, 98%, 99%) with the aminoacid sequence of SEQ ID NO: 26 or 674, or (iii) the amino acid sequenceof (i) or (ii) with one or more conservative amino acid modifications.

In some embodiments, at least one of the amino acids of the extension isacylated or alkylated. The amino acid comprising the acyl or alkyl groupmay be located at any position of extension of the analog. In certainembodiments, the acylated or alkylated amino acid of the extension islocated at one of positions 37, 38, 39, 40, 41, or 42 (according to thenumbering of SEQ ID NO: 1) of the analog. In certain embodiments, theacylated or alkylated amino acid is located at position 40 of theanalog.

In exemplary embodiments, the acyl or alkyl group is an acyl or alkylgroup which is non-native to a naturally-occurring amino acid. Forexample, the acyl or alkyl group may be a C4 to C30 (e.g., C12 to C18)fatty acyl group or C4 to C30 (e.g., C12 to C18) alkyl. The acyl oralkyl group may be any of those discussed herein.

In some embodiments, the acyl or alkyl group is attached directly to theamino acid, e.g., via the side chain of the amino acid. In otherembodiments, the acyl or alkyl group is attached to the amino acid via aspacer (e.g., an amino acid, a dipeptide, a tripeptide, a hydrophilicbifunctional spacer, a hydrophobic bifunctional spacer). In certainaspects, the spacer is 3 to 10 atoms in length. In some embodiments, thespacer is an amino acid or dipeptide comprising one or two of6-aminohexanoic acid, Ala, Pro, Leu, beta-Ala, gamma-Glu (e.g.,gamma-Glu-gamma-Glu). In particular aspects, the total length of thespacer is 14 to 28 atoms.

Also, in exemplary embodiments, the amino acid to which the acyl oralkyl group is attached may be any of those described herein, including,for example, an amino acid of Formula I, II, or III. The amino acidwhich is acylated or alkylated may be a Lys, for example. Suitable aminoacids comprising an acyl or alkyl group, as well as suitable acylgroups, alkyl groups, and spacers are described herein. See, forexample, the teachings under the sections entitled Acylation andAlkylation.

In other embodiments, 1-6 amino acids (e.g., 1-2, 1-3, 1-4, 1-5 aminoacids) of the extension are positive-charged amino acids, e.g., aminoacids of Formula IV, such as, for example, Lys. As used herein, the term“positive-charged amino acid” refers to any amino acid,naturally-occurring or non-naturally occurring, comprising a positivecharge on an atom of its side chain at a physiological pH. In certainaspects, the positive-charged amino acids are located at any ofpositions 37, 38, 39, 40, 41, 42, and 43. In specific embodiments, apositive-charged amino acid is located at position 40.

In other instances, the extension is acylated or alkylated as describedherein and comprises 1-6 positive charged amino acids as describedherein.

In yet other embodiments, the analogs which exhibit agonist activity atthe GIP receptor comprises (i) SEQ ID NO: 1 with at least one amino acidmodification, (ii) an extension of 1 to 21 amino acids (e.g., 5 to 18, 7to 15, 9 to 12 amino acids) C-terminal to the amino acid at position 29of the analog, and (iii) an amino acid comprising an acyl or alkyl groupwhich is non-native to a naturally-occurring amino acid which is locatedoutside of the C-terminal extension (e.g., at any of positions 1-29). Insome embodiments, the analog comprises an acylated or alkylated aminoacid at position 10. In particular aspects, the acyl or alkyl group is aC4 to C30 fatty acyl or C4 to C30 alkyl group. In some embodiments, theacyl or alkyl group is attached via a spacer, e.g., an amino acid,dipeptide, tripeptide, hydrophilic bifunctional spacer, hydrophobicbifunctional spacer). In certain aspects, the analog comprises an aminoacid modification which stabilizes the alpha helix, such as a saltbridge between a Glu at position 16 and a Lys at position 20, or analpha,alpha-disubstituted amino acid at any one, two, three, or more ofpositions 16, 20, 21, and 24. In specific aspects, the analogadditionally comprises amino acid modifications which confer DPP-IVprotease resistance, e.g., DMIA at position 1, AIB at position 2.Analogs comprising further amino acid modifications are contemplatedherein.

In certain embodiments, the analogs having GIP receptor activity exhibitat least 0.1% (e.g., at least 0.5%, 1%, 2%, 5%, 10%, 15%, or 20%)activity of native GIP at the GIP receptor when the analog lacks ahydrophilic moiety, e.g., PEG. In some embodiments, the analogs exhibitmore than 10%, (e.g., more than 20%, more than 50%, more than 75%, morethan 100%, more than 200%, more than 300%, more than 500%) activity ofnative GIP at the GIP receptor. In some embodiments, the analog exhibitsappreciable agonist activity at one or both of the GLP-1 and glucagonreceptors. In some aspects, the potency and/or selectivity for thesereceptors (GIP receptor and GLP-1 receptor and/or glucagon receptor) arewithin 1000-fold, 750-fold, 500-fold, 250-fold, or 100-fold (higher orlower). For example, the selectivity for the GLP-1 receptor of theanalogs having GIP receptor activity can be less than 1000-fold,500-fold, 100-fold, within 50-fold, within 25 fold, within 15 fold,within 10 fold) (higher or lower) the selectivity for the GIP receptorand/or the glucagon receptor.

In any of the aspects described herein, the invention may exclude any ofthe peptides disclosed in International Application Publication No. WO2010/011439, International Application Publication No. WO 2008/101017,or International Application Publication No. WO 2009/155258.

Uses

As described in detail in the Examples, the glucagon agonists of thepresent invention have enhanced biophysical stability and aqueoussolubility while demonstrating enhanced bioactivity relative to thenative peptide. Accordingly, the glucagon agonists of the presentinvention are believed to be suitable for any use that has previouslybeen described for the native glucagon peptide. Accordingly, themodified glucagon peptides described herein can be used to treathypoglycemia or to increase blood glucose level, to induce temporaryparalysis of the gut for radiological uses, or treat other metabolicdiseases that result from low blood levels of glucagon. The glucagonpeptides described herein also are expected to be used to reduce ormaintain body weight, or to treat hyperglycemia, or to reduce bloodglucose level, or to normalize blood glucose level.

The glucagon peptides of the invention may be administered alone or incombination with other anti-diabetic or anti-obesity agents.Anti-diabetic agents known in the art or under investigation includeinsulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide(Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide(Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride(Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide(Prandin) or nateglinide (Starlix); biguanides such as metformin(Glucophage) or phenformin; thiazolidinediones such as rosiglitazone(Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or otherPPARγ inhibitors; alpha glucosidase inhibitors that inhibit carbohydratedigestion, such as miglitol (Glyset), acarbose (Precose/Glucobay);exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4)inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependentglucose transporter 1) inhibitors; glucokinase activators (GKA);glucagon receptor antagonists (GRA); or FBPase (fructose1,6-bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phentermine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to XENICAL (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

One aspect of the present disclosure is directed to a pre-formulatedaqueous solution of the presently disclosed glucagon agonist for use intreating hypoglycemia. The improved stability and solubility of theagonist compositions described herein allow for the preparation ofpre-formulated aqueous solutions of glucagon for rapid administrationand treatment of hypoglycemia. In one embodiment a solution comprising apegylated glucagon agonist is provided for administration to a patientsuffering from hypoglycemia, wherein the total molecular weight of thePEG chains linked to the pegylated glucagon agonist is between about 500to about 5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide selected from the group consisting of SEQ ID NO: 23,SEQ ID NO: 24, and SEQ ID NO: 25, and glucagon agonist analogs of SEQ IDNO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or a pegylated lactamderivative of glucagon comprising the sequence of SEQ ID NO: 20, whereinthe side chain of an amino acid residue of said glucagon peptide iscovalently bound to the polyethylene glycol chain.

The treatment methods in accordance with the present invention,including but not limited to treatment of hypoglycemia, may comprise thesteps of administering the presently disclosed glucagon agonists to apatient using any standard route of administration, includingparenterally, such as intravenously, intraperitoneally, subcutaneouslyor intramuscularly, intrathecally, transdermally, rectally, orally,nasally or by inhalation. In one embodiment the composition isadministered subcutaneously or intramuscularly. In one embodiment, thecomposition is administered parenterally and the glucagon composition isprepackaged in a syringe. In another embodiment, the composition isprepackaged in an inhaler or other aerosolized drug delivery device.

Surprisingly, applicants have discovered that pegylated glucagonpeptides can be prepared that retain the parent peptide's bioactivityand specificity. However, increasing the length of the PEG chain, orattaching multiple PEG chains to the peptide, such that the totalmolecular weight of the linked PEG is greater than 5,000 Daltons, beginsto delay the time action of the modified glucagon. In accordance withone embodiment, a glucagon peptide of SEQ ID NO: 23, SEQ ID NO: 24, andSEQ ID NO: 25, or a glucagon agonist analog thereof, or a pegylatedlactam derivative of glucagon comprising the sequence of SEQ ID NO: 20is provided wherein the peptide comprises one or more polyethyleneglycol chains, wherein the total molecular weight of the linked PEG isgreater than 5,000 Daltons, and in one embodiment is greater than 10,000Daltons, but less than 40,000 Daltons. Such modified glucagon peptideshave a delayed or prolonged time of activity but without loss of thebioactivity. Accordingly, such compounds can be administered to extendthe effect of the administered glucagon peptide.

Glucagon peptides that have been modified to be covalently bound to aPEG chain having a molecular weight of greater than 10,000 Daltons canbe administered in conjunction with insulin to buffer the actions ofinsulin and help to maintain stable blood glucose levels in diabetics.The modified glucagon peptides of the present disclosure can beco-administered with insulin as a single composition, simultaneouslyadministered as separate solutions, or alternatively, the insulin andthe modified glucagon peptide can be administered at different timerelative to one another. In one embodiment the composition comprisinginsulin and the composition comprising the modified glucagon peptide areadministered within 12 hours of one another. The exact ratio of themodified glucagon peptide relative to the administered insulin will bedependent in part on determining the glucagon levels of the patient, andcan be determined through routine experimentation.

In accordance with one embodiment a composition is provided comprisinginsulin and a modified glucagon peptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 andglucagon agonist analogs thereof, wherein the modified glucagon peptidefurther comprises a polyethylene glycol chain covalently bound to anamino acid side chain at position 17, 21, 24 or 21 and 24. In oneembodiment the composition is an aqueous solution comprising insulin andthe glucagon analog. In embodiments where the glucagon peptide comprisesthe sequence of SEQ ID NO: 24 or SEQ ID NO: 25 the PEG chain iscovalently bound at position 21 or 24 of the glucagon peptide. In oneembodiment the polyethylene glycol chain has a molecular weight of about10,000 to about 40,000.

In accordance with one embodiment the modified glucagon peptidesdisclosed herein are used to induce temporary paralysis of theintestinal tract. This method has utility for radiological purposes andcomprises the step of administering an effective amount of apharmaceutical composition comprising a pegylated glucagon peptide, aglucagon peptide comprising a c-terminal extension or a dimer of suchpeptides. In one embodiment the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ IDNO: 14 and SEQ ID NO: 15. In one embodiment the glucagon peptide furthercomprises a PEG chain, of about 1,000 to 40,000 Daltons is covalentlybound to an amino acid residue at position 21 or 24. In one embodimentthe glucagon peptide is selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQID NO: 15. In one embodiment the PEG chain has a molecular weight ofabout 500 to about 5,000 Daltons.

In a further embodiment the composition used to induce temporaryparalysis of the intestinal tract comprises a first modified glucagonpeptide and a second modified glucagon peptide. The first modifiedpeptide comprises a sequence selected from the group consisting of SEQID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, optionally linked to a PEGchain of about 500 to about 5,000 Daltons, and the second peptidecomprises a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO: 24 and SEQ ID NO: 25, covalently linked to a PEG chain ofabout 10,000 to about 40,000 Daltons. In this embodiment the PEG chainof each peptide is covalently bound to an amino acid residue at eitherposition 17, 21 or 24 of the respective peptide, and independent of oneanother.

Oxyntomodulin, a naturally occurring digestive hormone found in thesmall intestine, has been reported to cause weight loss whenadministered to rats or humans (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e., SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 27 (KRNRNNIA). Accordingly,applicants believe that the bioactivity of oxyntomodulin can be retained(i.e., appetite suppression and induced weight loss/weight maintenance),while improving the solubility and stability of the compound andimproving the pharmacokinetics, by substituting the glucagon peptideportion of oxyntomodulin with the modified glucagon peptides disclosedherein. In addition applicants also believe that a truncatedOxyntomodulin molecule comprising a glucagon peptide of the invention,having the terminal four amino acids of oxyntomodulin removed will alsobe effective in suppressing appetite and inducing weight loss/weightmaintenance.

Accordingly, the present invention also encompasses the modifiedglucagon peptides of the present invention that have a carboxy terminalextension of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28. These compoundscan be administered to individuals to induce weight loss or preventweight gain. In accordance with one embodiment a glucagon agonist analogof SEQ ID NO: 33 or SEQ ID NO: 20, further comprising the amino acidsequence of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked to aminoacid 29 of the glucagon peptide, is administered to individuals toinduce weight loss or prevent weight gain. More particularly, theglucagon peptide comprises a sequence selected from the group consistingof SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14 and SEQ IDNO: 15, further comprising the amino acid sequence of SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28 linked to amino acid 29 of the glucagonpeptide.

Exendin-4, is a peptide made up of 39 amino acids. It is a powerfulstimulator of a receptor known as GLP-1. This peptide has also beenreported to suppress appetite and induce weight loss. Applicants havefound that the terminal sequence of Exendin-4 when added at the carboxyterminus of glucagon improves the solubility and stability of glucagonwithout compromising the bioactivity of glucagon. In one embodiment theterminal ten amino acids of Exendin-4 (i.e., the sequence of SEQ ID NO:26 (GPSSGAPPPS)) are linked to the carboxy terminus of a glucagonpeptide of the present disclosure. These fusion proteins are anticipatedto have pharmacological activity for suppressing appetite and inducingweight loss/weight maintenance. In accordance with one embodiment aglucagon agonist analog of SEQ ID NO: 33 or SEQ ID NO: 20, furthercomprising the amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS) or SEQID NO: 29 linked to amino acid 29 of the glucagon peptide, isadministered to individuals to induce weight loss or prevent weightgain. More particularly, the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO: 55 and SEQ ID NO: 56 further comprising the amino acidsequence of SEQ ID NO: 26 (GPSSGAPPPS) or SEQ ID NO: 29 linked to aminoacid 29 of the glucagon peptide. In one embodiment the administeredglucagon peptide analog comprises the sequence of SEQ ID NO: 64.

Multimers

The present disclosure also encompasses multimers of the modifiedglucagon peptides disclosed herein. Two or more of the modified glucagonpeptides can be linked together using standard linking agents andprocedures known to those skilled in the art. For example, dimers can beformed between two modified glucagon peptides through the use ofbifunctional thiol crosslinkers and bi-functional amine crosslinkers,particularly for the glucagon peptides that have been substituted withcysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues (e.g. SEQ ID NO: 3 and SEQ ID NO: 4). The dimer can be ahomodimer or alternatively can be a heterodimer. In certain embodiments,the linker connecting the two (or more) glucagon peptides is PEG, e.g.,a 5 kDa PEG, 20 kDa PEG. In some embodiments, the linker is a disulfidebond. For example, each monomer of the dimer may comprise a Cys residue(e.g., a terminal or internally positioned Cys) and the sulfur atom ofeach Cys residue participates in the formation of the disulfide bond. Insome aspects of the invention, the monomers are connected via terminalamino acids (e.g., N-terminal or C-terminal), via internal amino acids,or via a terminal amino acid of at least one monomer and an internalamino acid of at least one other monomer. In specific aspects, themonomers are not connected via an N-terminal amino acid. In someaspects, the monomers of the multimer are attached together in a“tail-to-tail” orientation in which the C-terminal amino acids of eachmonomer are attached together.

In one embodiment the dimer comprises a homodimer of a glucagon fusionpeptide wherein the glucagon peptide portion comprises SEQ ID NO: 11 orSEQ ID NO: 20 and an amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS),SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked to amino acid 29of the glucagon peptide. In another embodiment the dimer comprises ahomodimer of a glucagon agonist analog of SEQ ID NO: 11, wherein theglucagon peptide further comprises a polyethylene glycol chaincovalently bound to position 21 or 24 of the glucagon peptide.

In accordance with one embodiment a dimer is provided comprising a firstglucagon peptide bound to a second glucagon peptide via a linker,wherein the first glucagon peptide comprises a peptide selected from thegroup consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ IDNO: 11 and the second glucagon peptide comprises SEQ ID NO: 20. Inaccordance with another embodiment a dimer is provided comprising afirst glucagon peptide bound to a second glucagon peptide via a linker,wherein said first glucagon peptide comprises a sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and thesecond glucagon peptide comprise SEQ ID NO: 11, and pharmaceuticallyacceptable salts of said glucagon polypeptides. In accordance withanother embodiment a dimer is provided comprising a first glucagonpeptide bound to a second glucagon peptide via a linker, wherein saidfirst glucagon peptide is selected from the group consisting of SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and the second glucagonpeptide is independently selected from the group consisting of SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and pharmaceuticallyacceptable salts of said glucagon polypeptides. In one embodiment thefirst glucagon peptide is selected from the group consisting of SEQ IDNO: 20 and the second glucagon peptide is independently selected fromthe group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 11. Inone embodiment the dimer is formed between two peptides wherein eachpeptide comprises the amino acid sequence of SEQ ID NO: 11.

Kits

The modified glucagon peptides of the present invention can be providedin accordance with one embodiment as part of a kit. In one embodiment akit for administering a glucagon agonist to a patient in need thereof isprovided wherein the kit comprises a modified glucagon peptide selectedfrom the group consisting of 1) a glucagon peptide comprising thesequence of SEQ ID NO: 20, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:11;2) a glucagon fusion peptide comprising a glucagon agonist analog of SEQID NO: 11, SEQ ID NO: 20 or SEQ ID NO: 55, and an amino acid sequence ofSEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28(KRNR) linked to amino acid 29 of the glucagon peptide; and 3) apegylated glucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 51, furthercomprising an amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ IDNO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked to amino acid 29 of theglucagon peptide, wherein the PEG chain covalently bound to position 17,21 or 24 has a molecular weight of about 500 to about 40,000 Daltons. Inone embodiment the kit comprise a glucagon/GLP-1 co-agonist wherein thepeptide comprises a sequence selected from the group consisting of SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In one embodiment the kit is provided with a device for administeringthe glucagon composition to a patient, e.g. syringe needle, pen device,jet injector or other needle-free injector. The kit may alternatively orin addition include one or more containers, e.g., vials, tubes, bottles,single or multi-chambered pre-filled syringes, cartridges, infusionpumps (external or implantable), jet injectors, pre-filled pen devicesand the like, optionally containing the glucagon peptide in alyophilized form or in an aqueous solution. Preferably, the kits willalso include instructions for use. In accordance with one embodiment thedevice of the kit is an aerosol dispensing device, wherein thecomposition is prepackaged within the aerosol device. In anotherembodiment the kit comprises a syringe and a needle, and in oneembodiment the sterile glucagon composition is prepackaged within thesyringe.

Pharmaceutical Formulations

In accordance with one embodiment a pharmaceutical composition isprovided wherein the composition comprises a glucadon peptide of thepresent disclosure, or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The pharmaceutical composition cancomprise any pharmaceutically acceptable ingredient, including, forexample, acidifying agents, additives, adsorbents, aerosol propellants,air displacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers, filmforming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one ora combination of the following components: acacia, acesulfame potassium,acetyltributyl citrate, acetyltriethyl citrate, agar, albumin, alcohol,dehydrated alcohol, denatured alcohol, dilute alcohol, aleuritic acid,alginic acid, aliphatic polyesters, alumina, aluminum hydroxide,aluminum stearate, amylopectin, α-amylose, ascorbic acid, ascorbylpalmitate, aspartame, bacteriostatic water for injection, bentonite,bentonite magma, benzalkonium chloride, benzethonium chloride, benzoicacid, benzyl alcohol, benzyl benzoate, bronopol, butylatedhydroxyanisole, butylated hydroxytoluene, butylparaben, butylparabensodium, calcium alginate, calcium ascorbate, calcium carbonate, calciumcyclamate, dibasic anhydrous calcium phosphate, dibasic dehydratecalcium phosphate, tribasic calcium phosphate, calcium propionate,calcium silicate, calcium sorbate, calcium stearate, calcium sulfate,calcium sulfate hemihydrate, canola oil, carbomer, carbon dioxide,carboxymethyl cellulose calcium, carboxymethyl cellulose sodium,β-carotene, carrageenan, castor oil, hydrogenated castor oil, cationicemulsifying wax, cellulose acetate, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, silicifiedmicrocrystalline cellulose, sodium carboxymethyl cellulose, cetostearylalcohol, cetrimide, cetyl alcohol, chlorhexidine, chlorobutanol,chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidinegluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC),chlorodifluoromethane, chlorofluorocarbons (CFC) chlorophenoxyethanol,chloroxylenol, corn syrup solids, anhydrous citric acid, citric acidmonohydrate, cocoa butter, coloring agents, corn oil, cottonseed oil,cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium,crospovidone, cyclamic acid, cyclodextrins, dextrates, dextrin,dextrose, dextrose anhydrous, diazolidinyl urea, dibutyl phthalate,dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane(HFC), dimethyl-β-cyclodextrin, cyclodextrin-type compounds such asCaptisol®, dimethyl ether, dimethyl phthalate, dipotassium edentate,disodium edentate, disodium hydrogen phosphate, docusate calcium,docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

The pharmaceutical formulations disclosed herein may be designed to beshort-acting, fast-releasing, long-acting, or sustained-releasing asdescribed below. The pharmaceutical formulations may also be formulatedfor immediate release, controlled release or for slow release. Theinstant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. The disclosed pharmaceutical formulations may be administeredaccording to any regime including, for example, daily (1 time per day, 2times per day, 3 times per day, 4 times per day, 5 times per day, 6times per day), every two days, every three days, every four days, everyfive days, every six days, weekly, bi-weekly, every three weeks,monthly, or bi-monthly.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capabale of buffering at the desired pHsuch as, for example, phosphate buffers (e.g. PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g. at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Position 3 Modification

Any of the glucagon peptides, including glucagon analogs, glucagonagonist analogs, glucagon co-agonists, and glucagon/GLP-1 co-agonistmolecules, described herein may be modified to contain a modification atposition 3, e.g., Gln substituted with Glu, to produce a peptide withhigh selectivity, e.g., tenfold selectivity, for the GLP-1 receptor ascompared to the selectivity for the glucagon receptor.

Any of the glucagon peptides, including glucagon analogs, glucagonagonist analogs, glucagon co-agonists, and glucagon/GLP-1 co-agonistmolecules, described herein may be modified to contain a modification atposition 3, e.g., Gln substituted with a glutamine analog (e.g.Dab(Ac)), without a substantial loss of activity at the glucagonreceptor, and in some cases, with an enhancement of glucagon receptoractivity.

Preparation Methods

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

EXAMPLES General Synthesis Protocol

Glucagon analogs were synthesized using HBTU-activated “Fast Boc” singlecoupling starting from 0.2 mmole of Boc Thr(OBzl)Pam resin on a modifiedApplied Biosystem 430 A peptide synthesizer. Boc amino acids and HBTUwere obtained from Midwest Biotech (Fishers, Ind.). Side chainprotecting groups used were: Arg(Tos), Asn(Xan), Asp(OcHex),Cys(pMeBzl), His(Bom), Lys(2Cl—Z), Ser(OBzl), Thr(OBzl), Tyr(2Br—Z), andTrp(CHO). The side-chain protecting group on the N-terminal His was Boc.

Each completed peptidyl resin was treated with a solution of 20%piperidine in dimethylformamide to remove the formyl group from thetryptophan. Liquid hydrogen fluoride cleavages were performed in thepresence of p-cresol and dimethyl sulfide. The cleavage was run for 1hour in an ice bath using an HF apparatus (Penninsula Labs). Afterevaporation of the HF, the residue was suspended in diethyl ether andthe solid materials were filtered. Each peptide was extracted into 30-70ml aqueous acetic acid and a diluted aliquot was analyzed by HPLC[Beckman System Gold, 0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer=0.1% TFA, B=0.1% TFA/90% acetonitrile, gradient of 10% to 80% Bover 10 min].

Purification was done on a FPLC over a 2.2×25 cm Kromasil C18 columnwhile monitoring the UV at 214 nm and collecting 5 minute fractions. Thehomogeneous fractions were combined and lyophilized to give a productpurity of >95%. The correct molecular mass and purity were confirmedusing MALDI-mass spectral analysis.

General Pegylation Protocol: (Cys-maleimido)

Typically, the glucagon Cys analog is dissolved in phosphate bufferedsaline (5-10 mg/ml) and 0.01M ethylenediamine tetraacetic acid is added(10-15% of total volume). Excess (2-fold) maleimido methoxyPEG reagent(Nektar) is added and the reaction stirred at room temp while monitoringreaction progress by HPLC. After 8-24 hrs, the reaction mixture, isacidified and loaded onto a preparative reverse phase column forpurification using 0.1% TFA/acetonitrile gradient. The appropriatefractions were combined and lyophilized to give the desired pegylatedanalogs.

Example 1 Synthesis of Glucagon Cys¹⁷(1-29) and Similar MonoCys Analogs

0.2 mmole Boc Thr(OBzl) Pam resin (SynChem Inc) in a 60 ml reactionvessel and the following sequence was entered and run on a modifiedApplied Biosystems 430A Peptide Synthesizer using FastBoc HBTU-activatedsingle couplings.

HSQGTFTSDYSKYLDSCRAQDFVQWLMNT (SEQ ID NO: 35)The following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to an HF reaction vessel anddried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were addedalong with a magnetic stir bar. The vessel was attached to the HFapparatus (Pennisula Labs), cooled in a dry ice/methanol bath,evacuated, and aprox. 10 ml liquid hydrogen fluoride was condensed in.The reaction was stirred in an ice bath for 1 hr then the HF was removedin vacuo. The residue was suspended in ethyl ether; the solids werefiltered, washed with ether, and the peptide extracted into 50 mlaqueous acetic acid. An analytical HPLC was run [0.46×5 cm Zorbax C8, 1ml/min, 45 C, 214 nm, A buffer of 0.1% TFA, B buffer of 0.1% TFA/90%ACN, gradient=10% B to 80% B over 10 min.] with a small sample of thecleavage extract. The remaining extract was loaded onto a 2.2×25 cmKromasil C18 preparative reverse phase column and an acetonitrilegradient was run using a Pharmacia FPLC system. 5 min fractions werecollected while monitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1%TFA/50% acetonitrile. Gradient=30% B to 100% B over 450 min.

The fractions containing the purest product (48-52) were combinedfrozen, and lyophilized to give 30.1 mg. An HPLC analysis of the productdemonstrated a purity of >90% and MALDI mass spectral analysisdemonstrated the desired mass of 3429.7. Glucagon Cys²¹, Glucagon Cys²⁴,and Glucagon Cys²⁹ were similarly prepared.

Example 2 Synthesis of Glucagon-Cex and Other C-Terminal ExtendedAnalogs

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasplaced in a 60 ml reaction vessel and the following sequence was enteredand run on a modified Applied Biosystems 430A peptide synthesizer usingFastBoc HBTU-activated single couplings.

(SEQ ID NO: 36) HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPSThe following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to HF reaction vessel and driedin vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added alongwith a magnetic stir bar. The vessel was attached to the HF apparatus(Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, andaprox. 10 ml liquid hydrogen fluoride was condensed in. The reaction wasstirred in an ice bath for 1 hr then the HF was removed in vacuo. Theresidue was suspended in ethyl ether; the solids were filtered, washedwith ether, and the peptide extracted into 50 ml aqueous acetic acid. Ananalytical HPLC was run [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer of 0.1% TFA, B buffer of 0.1% TFA/90% ACN, gradient=10% B to 80%B over 10 min.] on an aliquot of the cleavage extract. The extract wasloaded onto a 2.2×25 cm Kromasil C18 preparative reverse phase columnand an acetonitrile gradient was run for elution using a Pharmacia FPLCsystem. 5 min fractions were collected while monitoring the UV at 214 nm(2.0 A). A=0.1% TFA, B=0.1% TFA/50% acetonitrile. Gradient=30% B to 100%B over 450 min. Fractions 58-65 were combined, frozen and lyophilized togive 198.1 mg.

HPLC analysis of the product showed a purity of greater than 95%. MALDImass spectral analysis showed the presence of the desired theoreticalmass of 4316.7 with the product as a C-terminal amide. Oxyntomodulin andoxyntomodulin-KRNR were similarly prepared as the C-terminal carboxylicacids starting with the appropriately loaded PAM-resin.

Example 3 Glucagon Cys¹⁷ Mal-PEG-SK

15.1 mg of Glucagon Cys¹⁷(1-29) and 27.3 mg methoxy poly(ethyleneglycol)maleimide avg. M.W.5000 (mPEG-Mal-5000, Nektar Therapeutics) weredissolved in 3.5 ml phosphate buffered saline (PBS) and 0.5 ml 0.01Methylenediamine tetraacetic acid (EDTA) was added. The reaction wasstirred at room temperature and the progress of the reaction wasmonitored by HPLC analysis [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm(0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10min.].

After 5 hours, the reaction mixture was loaded onto 2.2×25 cm KromasilC18 preparastive reverse phase column. An acetonitrile gradient was runon a Pharmacia FPLC while monitoring the UV wavelength at 214 nm andcollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% acetonitrile,gradient=30% B to 100% B over 450 min. The fractions corresponding tothe product were combined, frozen and lyophilized to give 25.9 mg.

This product was analyzed on HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C,214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% Bover 10 min.] which showed a purity of aprox. 90%. MALDI (matrixassisted laser desorption ionization) mass spectral analysis showed abroad mass range (typical of PEG derivatives) of 8700 to 9500. Thisshows an addition to the mass of the starting glucagon peptide (3429) ofapproximately 5,000 a.m.u.

Example 4 Glucagon Cys²¹ Mal-PEG-SK

21.6 mg of Glucagon Cys²¹(1-29) and 24 mg mPEG-MAL-5000 (NektarTherapeutics) were dissolved in 3.5 ml phosphate buffered saline (PBS)and 0.5 ml 0.01M ethylene diamine tetraacetic acid (EDTA) was added. Thereaction was stirred at room temp. After 2 hrs, another 12.7 mg ofmPEG-MAL-5000 was added. After 8 hrs, the reaction mixture was loadedonto a 2.2×25 cm Vydac C18 preparative reverse phase column and anacetonitrile gradient was run on a Pharmacia FPLC at 4 ml/min whilecollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% ACN. Gradient=20%to 80% B over 450 min.

The fractions corresponding to the appearance of product were combinedfrozen and lyophilized to give 34 mg. Analysis of the product byanalytical HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm (0.5 A),A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10 min.]showed a homogeneous product that was different than starting glucagonpeptide. MALDI (matrix assisted laser desorption ionization) massspectral analysis showed a broad mass range (typical of PEG analogs) of8700 to 9700. This shows an addition to the mass of the startingglucagon peptide (3470) of approximately 5,000 a.m.u.

Example 5 Glucagon Cys²⁴ Mal-PEG-SK

20.1 mg Glucagon C²⁴(1-29) and 39.5 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring and 0.5 ml0.01M EDTA was added. The reaction was stirred at room temp for 7 hrs,then another 40 mg of mPEG-Mal-5000 was added. After approximately 15hr, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was runusing a Pharmacia FPLC. 5 min. fractions were collected while monitoringthe UV at 214 nm (2.0 A). A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,gradient=30% B to 100% B over 450 min. The fractions corresponding toproduct were combined, frozen and lyophilized to give 45.8 mg. MALDImass spectral analysis showed a typical PEG broad signal with a maximumat 9175.2 which is approximately 5,000 a.m.u. more than Glucagon C²⁴(3457.8).

Example 6 Glucagon Cys²⁴ Mal-PEG-20K

25.7 mg of Glucagon Cys²⁴(1-29) and 40.7 mg mPEG-Mal-20K (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at room temp.and 0.5 ml 0.01M EDTA was added. After 6 hrs, the ratio of startingmaterial to product was aprox. 60:40 as determined by HPLC. Another 25.1mg of mPEG-Mal-20K was added and the reaction allowed to stir another 16hrs. The product ratio had not significantly improved, so the reactionmixture was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column and purified on a Pharmacia FPLC using a gradient of 30% Bto 100% B over 450 min. A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,flow=4 ml/min, and 5 min fractions were collected while monitoring theUV at 214 nm (2.0 A). The fractions containing homogeneous product werecombined, frozen and lyophilized to give 25.7 mg. Purity as determinedby analytical HPLC was ˜90%. A MALDI mass spectral analysis showed abroad peak from 23,000 to 27,000 which is approximately 20,000 a.m.u.more than starting Glucagon C²⁴ (3457.8).

Example 7 Glucagon Cys²⁹ Mal-PEG-SK

20.0 mg of Glucagon Cys²⁹(1-29) and 24.7 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at roomtemperature and 0.5 ml 0.01M EDTA was added. After 4 hr, another 15.6 mgof mPEG-Mal-5000 was added to drive the reaction to completion. After 8hrs, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was run ona Pharmacia FPLC system. 5 min fractions were collected while monitoringthe UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN. Fractions75-97 were combined frozen and lyophilized to give 40.0 mg of productthat is different than recovered starting material on HPLC (fractions58-63). Analysis of the product by analytical HPLC [0.46×5 cm Zorbax C8,1 ml/min, 45 C, 214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN,gradient=10% B to 80% B over 10 min.] showed a purity greater than 95%.MALDI mass spectral analysis showed the presence of a PEG component witha mass range of 8,000 to 10,000 (maximum at 9025.3) which is 5,540a.m.u. greater than starting material (3484.8).

Example 8 Glucagon Cys²⁴ (2-butyrolactone)

To 24.7 mg of Glucagon Cys²⁴(1-29) was added 4 ml 0.05M ammoniumbicarbonate/50% acetonitrile and 5.5 ul of a solution of2-bromo-4-hydroxybutyric acid-γ-lactone (100 ul in 900 ul acetonitrile).After 3 hrs of stirring at room temperature, another 105 ul of lactonesolution was added to the reaction mixture which was stirred another 15hrs. The reaction mixture was diluted to 10 ml with 10% aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column. An acetonitrile gradient (20% B to 80% B over 450 min) wasrun on a Pharmacia FPLC while collecting 5 min fractions and monitoringthe UV at 214 nm (2.0 A). Flow=4 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 74-77 were combined frozen and lyophilized to give 7.5 mg.HPLC analysis showed a purity of 95% and MALDI mass spect analysisshowed a mass of 3540.7 or 84 mass units more than starting material.This result is consistent with the addition of a single butyrolactonemoiety.

Example 9 Glucagon Cys²⁴(S-carboxymethyl)

18.1 mg of Glucagon Cys²⁴(1-29) was dissolved in 9.4 ml 0.1M sodiumphosphate buffer (pH=9.2) and 0.6 ml bromoacetic acid solution (1.3mg/ml in acetonitrile) was added. The reaction was stirred at roomtemperature and the reaction progress was followed by analytical HPLC.After 1 hr another 0.1 ml bromoacetic acid solution was added. Thereaction was stirred another 60 min. then acidified with aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column for purification. An acetonitrile gradient was run on aPharmacia FPLC (flow=4 ml/min) while collecting 5 min fractions andmonitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 26-29 were combined frozen and lyophilized to give several mgof product. Analytical HPLC showed a purity of 90% and MALDI massspectral analysis confirmed a mass of 3515 for the desired product.

Example 10

Glucagon Cys²⁴ maleimido, PEG-3.4K-dimer 16 mg Glucagon Cys²⁴ and 1.02mg Mal-PEG-Mal-3400, poly(ethyleneglycol)-bis-maleimide avg. M.W. 3400,(Nektar Therpeutics) were dissolved in 3.5 phosphate buffered saline and0.5 ml 0.01M EDTA and the reaction was stirred at room temperature.After 16 hrs, another 16 mg of Glucagon Cys²⁴ was added and the stirringcontinued. After approximately 40 hrs, the reaction mixture was loadedonto a Pharmcia PepRPC 16/10 column and an acetonitrile gradient was runon a Pharmacia FPLC while collecting 2 min fractions and monitoring theUV at 214 nm (2.0 A). Flow=2 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 69-74 were combined frozen and lyophilized to give 10.4 mg.Analytical HPLC showed a purity of 90% and MALDI mass spectral analysisshows a component in the 9500-11,000 range which is consistent with thedesired dimer.

Example 11 Synthesis of Glucagon Lactams

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasadded to a 60 mL reaction vessels and the following sequence wasassembled on a modified Applied Biosystems 430A peptide synthesizerusing Boc DEPBT-activated single couplings.

(12-16 Lactam; SEQ ID NO: 12) HSQGTFTSDYSKYLDERRAQDFVQWLMNT-NH2

The following side chain protecting groups were used: Arg(Tos),Asp(OcHx), Asn(Xan), Glu(OFm), His(BOM), Lys(Fmoc), Ser(Bzl), Thr(Bzl),Trp(CHO), Tyr(Br—Z). Lys(Cl—Z) was used at position 12 if lactams wereconstructed from 16-20, 20-24, or 24-28. The completed peptidyl resinwas treated with 20% piperidine/dimethylformamide for one hour withrotation to remove the Trp formyl group as well as the Fmoc and OFmprotection from Lys12 and Glu16. Upon confirmation of removal by apositive ninhydrin test, the resin was washed with dimethylformamide,followed by dichloromethane and than again with dimethylformamide. Theresin was treated with 520 mg (1 mmole)Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) in dimethylformamide and diisopropylethylamine (DIEA). Thereaction proceeded for 8-10 hours and the cyclization was confirmed by anegative ninhydrin reaction. The resin was washed withdimethylformamide, followed by dichloromethane and subsequently treatedwith trifluoroacetic acid for 10 minutes. The removal of the Boc groupwas confirmed by a positive ninhydrin reaction. The resin was washedwith dimethylformamide and dichloromethane and dried before beingtransferred to a hydrofluoric acid (HF) reaction vessel. 500 μl p-cresolwas added along with a magnetic stir bar. The vessel was attached to theHF apparatus (Peninsula Labs), cooled in a dry ice/methanol bath,evacuated, and approximately 10 mL of liquid hydrofluoric acid wascondensed into the vessel. The reaction was stirred for 1 hour in an icebath and the HF was subsequently removed in vacuo. The residue wassuspended in ethyl ether; the solids were filtered, washed with ether,and the peptide was solubilized with 150 mL 20% acetonitrile/1% aceticacid.

An analytical HPLC analysis of the crude solubilized peptide wasconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% TFA/10% ACN, B buffer 0.1% TFA/100% ACN,gradient 5-95% B over 15 minutes]. The extract was diluted twofold withwater and loaded onto a 2.2×25 cm Vydac C4 preparative reverse phasecolumn and eluted using an acetonitrile gradient on a Waters HPLC system(A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% CAN and agradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLCanalysis of the purified peptide demonstrated greater than 95% purityand electrospray ionization mass spectral analysis confirmed a mass of3506 Da for the 12-16 lactam. Lactams from 16-20, 20-24, and 24-28 wereprepared similarly.

Example 12 Glucagon Solubility Assays

A solution (1 mg/ml or 3 mg/ml) of glucagon (or an analog) is preparedin 0.01N HCl. 100 ul of stock solution is diluted to 1 ml with 0.01N HCland the UV absorbance (276 nm) is determined. The pH of the remainingstock solution is adjusted to pH7 using

200-250 ul 0.1M Na₂HPO₄ (pH9.2). The solution is allowed to standovernight at 4° C. then centrifuged. 100 ul of supernatant is thendiluted to 1 ml with 0.01N HCl, and the UV absorbance is determined (induplicate).

The initial absorbance reading is compensated for the increase in volumeand the following calculation is used to establish percent solubility:

${\frac{{Final}\mspace{14mu} {Absorbance}}{{Initial}\mspace{14mu} {Absorbance}} \times 100} = {{percent}\mspace{14mu} {soluble}}$

Results are shown in Table 1 wherein Glucagon-Cex represents wild typeglucagon (SEQ ID NO: 1) plus a carboxy terminal addition of SEQ ID NO:26 and Glucagon-Cex R¹² represents SEQ ID NO: 39.

TABLE 1 Solubility date for glucagon analogs Analog Percent SolubleGlucagon 16 Glucagon-Cex, R12 104 Glucagon-Cex 87 Oxyntomodulin 104Glucagon, Cys17PEG5K 94 Glucagon, Cys21PEG5K 105 Glucagon, Cys24PEG5K133

Example 13 Glucagon Receptor Binding Assay

The affinity of peptides to the glucagon receptor was measured in acompetition binding assay utilizing scintillation proximity assaytechnology. Serial 3-fold dilutions of the peptides made inscintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 MNaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clearbottom plate (Corning Inc., Acton, Mass.) with 0.05 nM(3-[¹²⁵I]-iodotyrosyl) Tyr10 glucagon (Amersham Biosciences, Piscataway,N.J.), 1-6 micrograms per well, plasma membrane fragments prepared fromcells over-expressing human glucagon receptor, and 1 mg/wellpolyethyleneimine-treated wheat germ agglutinin type A scintillationproximity assay beads (Amersham Biosciences, Piscataway, N.J.). Upon 5min shaking at 800 rpm on a rotary shaker, the plate was incubated 12 hat room temperature and then read on MicroBeta1450 liquid scintillationcounter (Perkin-Elmer, Wellesley, Mass.). Non-specifically bound (NSB)radioactivity was measured in the wells with 4 times greaterconcentration of “cold” native ligand than the highest concentration intest samples and total bound radioactivity was detected in the wellswith no competitor. Percent specific binding was calculated asfollowing: % Specific Binding=((Bound-NSB)/(Total bound-NSB))×100. IC₅₀values were determined by using Origin software (OriginLab, Northampton,Mass.).

Example 14 Functional Assay-cAMP Synthesis

The ability of glucagon analogs to induce cAMP was measured in a fireflyluciferase-based reporter assay. HEK293 cells co-transfected with eitherglucagon- or GLP-1 receptor and luciferase gene linked to cAMPresponsive element were serum deprived by culturing 16 h in DMEM(Invitrogen, Carlsbad, Calif.) supplemented with 0.25% Bovine GrowthSerum (HyClone, Logan, Utah) and then incubated with serial dilutions ofeither glucagon, GLP-1 or novel glucagon analogs for 5 h at 37° C., 5%CO₂ in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation 100 microliters ofLucLite luminescence substrate reagent (Perkin-Elmer, Wellesley, Mass.)were added to each well. The plate was shaken briefly, incubated 10 minin the dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). Effective 50%concentrations were calculated by using Origin software (OriginLab,Northampton, Mass. Results are shown in FIGS. 3-9 and in Tables 2through 10.

TABLE 2 cAMP Induction by Glucagon Analogs with C-Terminus ExtensioncAMP Induction Glucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N*EC₅₀, nM N Glucagon 0.22 ± 0.09 14 3.85 ± 1.64 10 GLP-1 2214.00 ±182.43  2 0.04 ± 0.01 14 Glucagon Cex 0.25 ± 0.15 6 2.75 ± 2.03 7Oxyntomodulin 3.25 ± 1.65 5 2.53 ± 1.74 5 Oxyntomodulin KRNR 2.77 ± 1.744 3.21 ± 0.49 2 Glucagon R12 0.41 ± 0.17 6 0.48 ± 0.11 5 Glucagon R12Cex 0.35 ± 0.23 10 1.25 ± 0.63 10 Glucagon R12 K20 0.84 ± 0.40 5 0.82 ±0.49 5 Glucagon R12 K24 1.00 ± 0.39 4 1.25 ± 0.97 5 Glucagon R12 K290.81 ± 0.49 5 0.41 ± 0.24 6 Glucagon Amide 0.26 ± 0.15 3 1.90 ± 0.35 2Oxyntomodulin C24 2.54 ± 0.63 2 5.27 ± 0.26 2 Oxyntomodulin C24 PEG 0.97± 0.04 1 1.29 ± 0.11 1 20K *number of experiments

TABLE 3 cAMP Induction by Pegylated Glucagon Analogs cAMP InductionGlucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N* EC₅₀, nM N Glucagon0.33 ± 0.23 18 12.71 ± 3.74 2 Glucagon C17 PEG 5K 0.82 ± 0.15 4 55.86 ±1.13 2 Glucagon C21 PEG 5K 0.37 ± 0.16 6 11.52 ± 3.68 2 Glucagon C24 PEG5K 0.22 ± 0.10 12 13.65 ± 2.95 4 Glucagon C29 PEG 5K 0.96 ± 0.07 2 12.71± 3.74 2 Glucagon C24 PEG 20K 0.08 ± 0.05 3 Not determined Glucagon C24Dimer 0.10 ± 0.05 3 Not determined GLP-1 >1000  0.05 ± 0.02 4 *number ofexperiments

TABLE 4 cAMP Induction by E16 Glucagon Analogs Percent Potency Relativeto Native Ligand Peptide GRec GLP-1Rec E16 Gluc-NH2 187.2 17.8 Glucagon100.0 0.8 Gluc-NH2 43.2 4.0 NLeu3, E16 Gluc-NH2 7.6 20.6 E3, E16Gluc-NH2 1.6 28.8 Orn3, E16 Gluc-NH2 0.5 0.1 GLP-1 <0.1 100

TABLE 5 cAMP Induction by E16 Glucagon Analogs Percent Potency Relativeto Native Ligand Peptide GRec GLP-1Rec E16 Gluc-NH2 187.2 17.8 E15, E16Gluc-NH2 147.0 9.2 E16, K20 Gluc-NH2 130.1 41.5 Gluc-NH2 43.2 4.0

TABLE 6 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor Peptide EC50 (nM) StDev n EC50 (nM) StDev nGlucagon 0.28 0.14 10 4.51 N/A  1 Glucagon-NH2 0.53 0.33  8 1.82 0.96  5E16 Gluc-NH2 0.07 0.07 10 0.16 0.14 10 E16, G30 Gluc-NH2 0.41 0.36  50.24 0.10  5 E16, G30 Cluc-Cex 0.51 0.46  5 1.19 0.86  5 GLP-1 2214 N/A 1 0.03 0.02  9

TABLE 7 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor Peptide EC50 (nM) StDev n EC50(nM) StDev n E16Glucagon NH2 0.07 0.07 10 0.16 0.14 10 hCSO₃16 0.25 0.12  2 0.19 0.02  2Glucagon-NH2 hE16 Glucagon-NH2 0.17 0.08  2 0.25 0.03  2 H16Glucagon-NH2 0.45 0.3   2 0.38 0.11  2 Q16 Glucagon-NH2 0.22 0.1   20.39 0.08  2 D16 Glucagon-NH2 0.56 0.15  2 0.93 0.28  2 (S16)Glucagon-NH2 0.53 0.33  8 1.82 0.96  5

TABLE 8 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor Peptide EC50 (nM) StDev n EC50(nM) StDev n E16Glucagon NH2 0.07 0.07 10 0.16  0.14 10 T16 Glucagon NH2 0.10 0.02  31.999 0.48  3 G16 Glucagon NH2 0.10 0.01  3 2.46  0.60  3 Glucagon NH20.53 0.33  4 1.82  0.96  5 GLP-1 2214 N/A  1 0.03  0.02  9 E16 Gluc NH₂was 4-fold more potent at the glucagon receptor relative to G16-COOH andT16 Gluc NH₂, when the compounds were tested side by side.

TABLE 9 cAMP Induction by E16/Lactam Glucagon Analogs Percent PotencyRelative to Native Ligand Peptide GRec GLP-1Rec E24K28 Gluc-NH2 Lac196.4 12.5 E16K20 Gluc-NH2 Lac 180.8 63.0 K12E16 Gluc-NH2 Lac 154.2 63.3K20E24 Gluc-NH2 Lac 120.2 8.1 E16 Gluc-NH2 187.2 17.8 E16, K20 Gluc-NH2130.1 41.5 Glucagon 100.0 0.8 Gluc-NH2 43.2 4.0

TABLE 10 cAMP Induction by GLP-1 17-26 Glucagon Analogs Glucagon GLP-1Receptor Receptor EC50 EC50 Peptide (nM) StD (nM) StD GLP-1 0.023 0.002Gluc-NH2 0.159 0.023 E16 GLP-1 0.009 0.000 E16 Glucagon-NH2 0.072 0.007E16 GLP(17-26)Glu(27-29)-NH2 0.076 0.004 0.014 0.001 E16 GLP(17-29)-NH20.46 0.023 0.010 0.000 E16 GLP(17-29)-NH2 E24, K28 0.23 0.020 0.007 E16GLP(17-29)-NH2 E24, K28 Lactam 0.16 0.017 0.007 0.000

Example 15 Stability Assay for Glucagon Cys-maleimido PEG Analogs

Each glucagon analog was dissolved in water or PBS and an initial HPLCanalysis was conducted. After adjusting the pH (4, 5, 6, 7), the sampleswere incubated over a specified time period at 37° C. and re-analyzed byHPLC to determine the integrity of the peptide. The concentration of thespecific peptide of interest was determined and the percent remainingintact was calculated relative to the initial analysis. Results forGlucagon Cys²¹-maleimidoPEG_(5K) are shown in FIGS. 1 and 2.

Example 16

The following glucagon peptides are constructed generally as describedabove in Examples 1-11:

In all of the following sequences, “a” means a C-terminal amide.

(SEQ ID NO: 70) HSQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 71)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 72)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 73)HSQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 74)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 75)HSQGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 76)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 77)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 78)HSQGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 79)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 80)HSQGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 81)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 82)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 83)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 84)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 85)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 86)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (SEQ ID NO: 87)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 88)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 89)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 90)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 91)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 92)X1SQGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 93)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 94)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 95)X1SQGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 96)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 97)X1SQGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 98)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 99)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 100)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 101)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 102)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 103)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences, X1=(Des-amino)His

(SEQ ID NO: 104) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 105)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 106)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 107)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 108)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 109)HX2QGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 110)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 111)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 112)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 113)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 114)HX2QGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 115)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 116)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 117)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 118)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 119)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 120)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 121) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 122)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 123)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 124)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 125)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 126)HX2QGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 127)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 128)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 129)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 130)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 131)HX2QGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 132)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 133)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 134)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 135)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 136)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 137)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 138) HSEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 139)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 140)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 141)HSEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 142)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 143)HSEGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 144)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 145)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 146)HSEGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 147)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 148)HSEGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 149)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 150)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 151)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 152)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 153)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 154)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (SEQ ID NO: 155)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 156)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 157)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 158)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 159)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 160)X1SEGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 161)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 162)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 163)X1SEGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 164)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 165)X1SEGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 166)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 167)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 168)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 169)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 170)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 171)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 172) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 173)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 174)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 175)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 176)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 177)HX2EGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 178)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 179)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 180)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 181)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 182)HX2EGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 183)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 184)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 185)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 186)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 187)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 188)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 189) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 190)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 191)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 192)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 193)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 194)HX2EGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 195)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 196)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 197)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 198)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 199)HX2EGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 200)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 201)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 202)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 203)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 204)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 205)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 206) HSQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 207)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 208)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 209)HSQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 210)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 211)HSQGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 212)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 213)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 214)HSQGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 215)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 216)HSQGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 217)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 218)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 219)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 220)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 221)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 222)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (SEQ ID NO: 223)X1SQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 224)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 225)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 226)X1SQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 227)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 228)X1SQGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 229)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 230)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 231)X1SQGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 232)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 233)X1SQGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 234)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 235)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 236)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 237)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 238)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 239)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X1=(Des-amino)His; and wherein the C*is a Cys, or a Cys attached to a hydrophilic polymer, or alternativelythe C* is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 240) HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 241)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 242)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 243)HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 244)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 245)HX2QGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 246)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 247)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 248)HX2QGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 249)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 250)HX2QGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 251)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 252)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 253)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 254)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 255)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 256)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid; and whereinthe C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 kD average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(SEQ ID NO: 257) HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 258)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 259)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 260)HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 261)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 262)HX2QGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 263)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 264)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 265)HX2QGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 266)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 267)HX2QGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 268)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 269)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 270)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 271)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 272)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 273)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=(D-Ala); and wherein the C* is aCys, or a Cys attached to a hydrophilic polymer, or alternatively the C*is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 274) HSEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 275)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 276)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 277)HSEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 278)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 279)HSEGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 280)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 281)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 282)HSEGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 283)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 284)HSEGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 285)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 286)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 287)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 288)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 289)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 290)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (SEQ ID NO: 291)X1SEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 292)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 293)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 294)X1SEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 295)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 296)X1SEGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 297)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 298)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 299)X1SEGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 300)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 301)X1SEGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 302)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 303)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 304)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 305)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 306)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 307)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X1=(Des-amino)His; and wherein the C*is a Cys, or a Cys attached to a hydrophilic polymer, or alternativelythe C* is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 308) HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 309)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 310)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 311)HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 312)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 313)HX2EGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 314)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 315)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 316)HX2EGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 317)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 318)HX2EGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 319)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 320)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 321)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 322)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 323)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 324)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid; and whereinthe C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 kD average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(SEQ ID NO: 325) HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 326)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 327)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 328)HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 329)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 330)HX2EGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 331)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 332)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 333)HX2EGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 334)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 335)HX2EGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 336)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 337)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 338)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 339)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 340)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 341)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=(D-Ala); and wherein the C* is aCys, or a Cys attached to a hydrophilic polymer, or alternatively the C*is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 342) HSQGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 343)HSQGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 344)HSQGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 345)HSQGT FTSDY SKYLD C*QAAK EFIAW LVKGa (SEQ ID NO: 346)X1SQGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 347)X1SQGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 348)X1SQGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 349)X1SQGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X1=(Des-amino)His; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 350) HX2QGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 351)HX2QGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 352)HX2QGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 353)HX2QGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=Aminoisobutyric acid; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 354) HX2QGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 355)HX2QGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 356)HX2QGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 357)HX2QGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 358) HSEGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 359)HSEGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 360)HSEGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 361)HSEGT FTSDY SKYLD C*QAAK EFIAW LVKGa (SEQ ID NO: 362)X1SEGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 363)X1SEGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 364)X1SEGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 365)X1SEGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X1=(Des-amino)His; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 366) HX2EGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 367)HX2EGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 368)HX2EGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 369)HX2EGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 370) HX2EGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 371)HX2EGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 372)HX2EGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 373)HX2EGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 374) HSQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 375)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 376)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 377)HSQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 378)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 379)HSQGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 380)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 381)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 382)HSQGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 383)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 384)HSQGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 385)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 386)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 387)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (SEQ ID NO: 388)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 389)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 390)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 391)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 392)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 393)X1SQGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 394)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 395)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 396)X1SQGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 397)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 398)X1SQGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 399)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 400)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 401)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 402) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 403)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 404)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 405)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 406)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 407)HX2QGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 408)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 409)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 410)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 411)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 412)HX2QGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 413)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 414)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 415)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 416) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 417)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 418)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 419)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 420)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 421)HX2QGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 422)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 423)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 424)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 425)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 426)HX2QGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 427)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 428)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 429)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 430) HSEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 431)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 432)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 433)HSEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 434)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 435)HSEGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 436)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 437)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 438)HSEGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 439)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 440)HSEGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 441)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 442)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 443)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (SEQ ID NO: 444)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 445)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 446)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 447)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 448)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 449)X1SEGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 450)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 451)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 452)X1SEGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 453)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 454)X1SEGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 455)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 456)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 457)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 458) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 459)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 460)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 461)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 462)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 463)HX2EGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 464)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 465)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 466)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 467)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 468)HX2EGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 469)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 470)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 471)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 472) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 473)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 474)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 475)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 476)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 477)HX2EGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 478)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 479)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 480)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 481)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 482)HX2EGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 483)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 484)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 485)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=(D-Ala)The following glucagon peptides with a GLP-1/glucagon activity ratio ofabout 5 or more are also constructed generally as described above inExamples 1-11. Generally, in these peptides, AIB at position 2 providesDPP IV resistance but also significantly reduces glucagon activity.

(SEQ ID NO: 486) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa  (SEQ ID NO: 487)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNC*a  (SEQ ID NO: 488)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA   PPPSC*a (lactam @16-20; SEQ ID NO: 489) HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA  PPPSC*a (SEQ ID NO: 490) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSa  (lactam @ 16-20; SEQ ID NO: 491)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA   PPPSaWherein in the preceding sequences X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 492) HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (SEQ ID NO: 493)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNC*a (SEQ ID NO: 494)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 495)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNGG PSSGA PPPSC*a (SEQ ID NO: 496)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 497) HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNGG PSSGA PPPSa(SEQ ID NO: 498) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (SEQ ID NO: 499)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNC*a (SEQ ID NO: 500)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 501)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGA PPPSC*a (SEQ ID NO: 502)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 503) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSaWherein in the preceding sequences X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.The following glucagon peptides which are GLP-1/glucagon co-agonists arealso constructed generally as described above in Examples 1-11.Formation of a lactam bridge between amino acids 16 and 20 restores thereduction in glucagon activity caused by the substitution at position 2.

(lactam @ 16-20; SEQ ID NO: 504) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTaWherein in the preceding sequence X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(lactam @ 16-20; SEQ ID NO: 505) X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa(lactam @ 16-20; SEQ ID NO: 506) X1SQGT FTSDY SKYLD EQAAK EFIAW LMNC*a(lactam @ 16-20; SEQ ID NO: 507)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 508)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 509) X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSa(lactam @ 16-20; SEQ ID NO: 510) X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa(lactam @ 16-20; SEQ ID NO: 511) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa(lactam @ 16-20; SEQ ID NO: 512) X1SQGT FTSDY SKYLD ERRAK DFVQW LMNC*a(lactam @ 16-20; SEQ ID NO: 513)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSaWherein in the preceding sequences X1=DMIA (alpha,alpha-dimethylimidiazole acetic acid), and wherein the C* is a Cys, or a Cys attachedto a hydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(optionally with lactam @ 16-20; SEQ ID NO: 514)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTaWherein the C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 kD average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(lactam @ 16-20; SEQ ID NO: 517) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa(lactam @ 16-20; SEQ ID NO: 528) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa(SEQ ID NO: 531) HX2QGT FTSDY SKYLD ERRAK EFIC*W LMNGG PSSGA PPPSC*a(SEQ ID NO: 532) HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA PPPSC*C*a(SEQ ID NO: 533) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSaWherein in the preceding sequence X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 518) HSQGT FTSDYSKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 519)X1SQGT FTSDYSKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 520)X1SQGT FTSDYSKYLD EQAAK EFIAW LMNC*a (SEQ ID NO: 529)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSa (SEQ ID NO: 530)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTaWherein in the preceding sequences X1=DMIA (alpha, alpha-dimethylimidiazole acetic acid), and wherein the C* is a Cys, or a Cys attachedto a hydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 521) HSQGT FTSDYSKYLD SRRAQ DFVQW LMNTGPSSGAPPPSa(SEQ ID NO: 522) HSQGT FTSDYSKYLD SRRAQ DFVQW LMNGGPSSGAPPPSa(SEQ ID NO: 523) HSQGT FTSDYSKYLD SRRAQ DFVQW LMKGGPSSGAPPPSa(SEQ ID NO: 524) HSQGT FTSDYSKYLD SRRAQ DFVQW LVKGGPSSGAPPPSa(SEQ ID NO: 525) HSQGT FTSDYSKYLD SRRAQ DFVQW LMDGGPSSGAPPPSa(SEQ ID NO: 526) HSQGT FTSDYSKYLD ERRAK DFVQW LMDGGPSSGAPPPSa(SEQ ID NO: 527) HAEGT FTSDV SSYLE GQAAK EFIAW LVKGGa (SEQ ID NO: 61)X1X2QGT FTSDY SKYLD ERX5AK DFVX3W LMNX4whereinX1=His, D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine or alpha,alpha-dimethyl imidiazoleacetic acid (DMIA) N-methyl histidine, alpha-methyl histidine, orimidazole acetic acid,X2=Ser, D-serine, Ala, Val, glycine, N-methyl serine or aminoisobutyricacid (AIB), N-methyl alanine and D-alanine.

X3=Ala, Gln or Cys-PEG X4=Thr-CONH2 or Cys-PEG or GGPSSGAPPPS (SEQ IDNO: 515) or GGPSSGAPPPSC-PEG (SEQ ID NO: 516)

Provided that when X3 is Cys-PEG, X4 is not Cys-PEG or GGPSSGAPPPSC-PEG(SEQ ID NO: 516), and when X2=Ser, X1 is not His.

X5=Ala or Arg

(SEQ ID NO: 62) X1X2QGT FTSDY SKYLD EQ X5AK EFI X3W LMNX4whereinX1=His, D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine or alpha,alpha-dimethyl imidiazoleacetic acid (DMIA), N-methyl histidine, alpha-methyl histidine, orimidazole acetic acidX2=Ser, D-serine, Ala, Val, glycine, N-methyl serine or aminoisobutyricacid (AIB), N-methyl alanine and D-alanine.

X3=Ala, Gln or Cys-PEG X4=Thr-CONH2 or Cys-PEG or GGPSSGAPPPS (SEQ IDNO: 515) or GGPSSGAPPPSC-PEG (SEQ ID NO: 516)

Provided that when X3 is Cys-PEG, X4 is not Cys-PEG or GGPSSGAPPPSC-PEG(SEQ ID NO: 516), and when X2=Ser, X1 is not His.

X5=Ala or Arg

HSEGT FTSDY SKYLD EQAAK EFIAW LXNTa (SEQ ID NO: 554), wherein X atposition 27 is Norleucine, wherein the amino acid at position 29 isamidated

Any of the preceding sequences can include additional modifications,e.g., 1, 2, 3, 4 or 5 modifications that do not destroy activity,including but not limited to W10 or R20 substitutions that can be usedto enhance potency. Any of the preceding sequences can also be producedwithout the modifications that confer DPP IV resistance, i.e., in whichthe native His is at position 1 and the native Ser is at position 2. Inaddition, any of the preceding compounds may optionally be linked to aconjugate, such as a heterologous polypeptide, an immunoglobulin or aportion thereof (e.g. Fc region), a targeting agent, a diagnostic label,or a diagnostic or therapeutic agent.

Example 17

The following glucagon peptides modified to comprise the c-terminalextension of SEQ ID NO: 26 linked to the carboxy terminus of theglucagon peptide were constructed generally as described above inExamples 1-11 and assayed for activity at the GLP-1 and glucagonreceptors using the in vitro assay described in Example 14.

Table 11 represents the activity of various glucagon analogs at theglucagon and GLP-1 receptors. The data shows that for glucagon analogscomprising the c-terminal extension of SEQ ID NO: 26, amino acidsubstitutions at positions 16, 20, 28 and 29 can impact the analogsactivity at the GLP-1 receptor.

TABLE 11 Glucagon-Cex Structure Activity Relationship Glucagon ReceptorGLP-1 Receptor EC50 Relative Relative Glucagon Peptide (nM) Potency (%)EC50 (nM) Potency (%) MNT²⁹  0.086 100 (SEQ ID NO: 1) MNTG³⁰ PSSGAPPPS0.14 61 1.19 2 (SEQ ID NO: 521) MNGG³⁰ PSSGAPPPS 0.28 30 0.31 8(SEQ ID NO: 522) MKGG³⁰ PSSGAPPPS 0.61 14 0.80 3 (SEQ ID NO: 523)VKGG³⁰ PSSGAPPPS 1.16 7 0.21 12 (SEQ ID NO: 524) MDGG³⁰ PSSGAPPPS 0.1272 0.13 19 (SEQ ID NO: 525) E¹⁶K²⁰-MDGG³⁰ PSSGAPPPS 0.22 39 0.020 125(SEQ ID NO: 526) GLP-1-VKGG³⁰ 0.025 100 (SEQ ID NO: 527)

Example 18

Table 12 represents in vitro data accumulated for various glucagonpeptides comparing their relative activities at the glucagon and GLP-1receptors.

TABLE 12 COMPARISON OF AGONISTS AND CO-AGONISTS w/ and w/o PEG % PotencyRelative to Native CONTROLS GR GL-1R Glucagon 100 0.78 GLP-1 <0.01 100Parent w/o PEG Parent w/PEG % Potency % Potency Relative to Relative toNative Native GR GLP-1R GR GLP-1R AGONISTS Chimera AIB2, Cys24 (SEQ IDNO: 486) 15.4 160.6 2.6 82.5 Chimera AIB2, Cys29 (SEQ ID NO: 487) 20.1124.6 5.6 54.3 Chimera AIB2, Gly29,30 Cys40 Cex (SEQ ID NO: 2.2 359.10.3 68.8 488) Chimera AIB2, Gly29,30 Cys40 Cex Lactam (SEQ ID 14.2 169.63.2 63.6 NO: 489) Chimera AIB2, Gly29,30 Cys24 Cex (SEQ ID NO: 2.5 457.80.2 95.4 490) Chimera AIB2, Gly29,30 Cys24 Cex Lactam (SEQ ID 25.2 381.51.4 96.4 NO: 491) E16, K20AIB2, A18 Cys24 (SEQ ID NO: 492) — — 1.1 73.5E16, K20AIB2, A18 Gly29,30 Cys24 Cex (SEQ ID — — 0.1 88.5 NO: 496)CO-AGONISTS Chimera DMIA1, Cys24 Lactam (SEQ ID NO: 505) 160.7 82.5 19.112.5 Chimera AIB2, Cys24 Lactam (SEQ ID NO: 504) 114.2 230.4 9.2 38.0Chimera DMIA1, Cys29 Lactam (SEQ ID NO: 506) — — — — Chimera DMIA1,Gly29,30 Cys40 Cex Lactam (SEQ — — — — ID NO: 507) E16, K20 DMIA1,Gly29,30 Cys40 Cex — — — — Lactam (SEQ ID NO: 508) Chimera DMIA1,Gly29,30 Cys24 Cex Lactam (SEQ — — — — ID NO: 509) E16, K20 DMIA1, Cys24Lactam (SEQ ID NO: 510) — — 64.1 9.3 E16, K20 AIB2, Cys24 Lactam (SEQ IDNO: 517) 108.3 96.9 15.8 31.0 Chimera Cys24 (SEQ ID NO: 518) — — 19.829.3 E16, K20 DMIA1, Gly29,30 Cys24 Cex 116.0 78.3 12.6 11.3 Lactam (SEQID NO: 513) Chimera DMIA1, Cys29 (SEQ ID NO: 520) — — 5.3 27.3 ChimeraDMIA1, Cys24 (SEQ ID NO: 519) 28.9 64.5 6.9 19.3

Example 19

Acylated and/or PEGylated peptides were prepared as follows. Peptideswere synthesized on a solid support resin using either a CS Bio 4886Peptide Synthesizer or Applied Biosystems 430A Peptide Synthesizer. Insitu neutralization chemistry was used as described by Schnolzer et al.,Int. J. Peptide Protein Res. 40: 180-193 (1992). For acylated peptides,the target amino acid residue to be acylated (e.g., position ten) wassubstituted with an Nε-FMOC lysine residue. Treatment of the completedN-terminally BOC protected peptide with 20% piperidine in DMF for 30minutes removed FMOC/formyl groups. Coupling to the free ε-amino Lysresidue was achieved by coupling a ten-fold molar excess of either anFMOC-protected spacer amino acid (ex. FMOC-(N-BOC)-Tryptophan-OH) oracyl chain (ex. C17-COOH) and PyBOP or DEPBT coupling reagent inDMF/DIEA. Subsequent removal of the spacer amino acid's FMOC group isfollowed by repetition of coupling with an acyl chain. Final treatmentwith 100% TFA resulted in removal of any side chain protecting groupsand the N-terminal BOC group. Peptide resins were neutralized with 5%DIEA/DMF, dried, and then cleaved from the support using HF/p-cresol,95:5, at 0° C. for one hour. Following ether extraction, a 5% HOAcsolution was used to solvate the crude peptide. A sample of the solutionwas then verified to contain the correct molecular weight peptide byESI-MS. Correct peptides were purified by RP-HPLC using a lineargradient of 10% CH3CN/0.1% TFA to 0.1% TFA in 100% CH3CN. A Vydac C18 22mm×250 mm protein column was used for the purification. Acylated peptideanalogs generally completed elution by a buffer ratio of 20:80. Portionswere pooled together and checked for purity on an analytical RP-HPLC.Pure fractions were lyophilized yielding white, solid peptides. Yieldstypically ranged from 10 mg to 100 mg depending on the synthesis.

If a peptide comprises a lactam bridge and target residues to beacylated, acylation is carried out as described above upon addition ofthat amino acid to the peptide backbone.

For peptide pegylation, 40 kDa methoxy poly(ethylene glycol)maleimido-propionamide (Chirotech Technology Ltd.) was reacted with amolar equivalent of peptide in 7M Urea, 50 mM Tris-HCl buffer using theminimal amount of solvent needed to dissolve both peptide and PEG into aclear solution (generally less than 2 mL for a reaction using 2-3 mgpeptide). Vigorous stirring at room temperature commenced for 4-6 hoursand the reaction analyzed by analytical RP-HPLC. PEGylated productsappeared distinctly from the starting material with decreased retentiontimes. Purification was performed on a Vydac C4 column with conditionssimilar to those used for the initial peptide purification. Elutionoccurred around buffer ratios of 50:50. Fractions of pure PEGylatedpeptide were found and lyophilized. Yields were above 50%, varying perreaction.

Peptides were assayed for biological activity, by co-tranfecting HEK293cells with either the glucagon receptor (GLUR) or GLP-1 receptor(GLP-1R) and a luciferase gene linked to a cAMP responsive element. Thetransfected cells were serum deprived by culturing for 16 hours in DMEMsupplemented with 0.25% Bovine Growth Serum and then incubated for 5hours with serial dilutions of the selected analogs and either Glucagonor GLP-1 as standards, respectively. Peptide absorbance readings wereobtained from UV Absorbance measurements at 280 nm on a Genesys 6Spectrophotometer (Thermo Electron Corporation). Beer's Law was used tocalculate solution concentrations based on the number of tryptophan andtyrosine residues in each analog. At the end of the incubation, 100 μLLucLite luminescence substrate reagent was added to each well, the platesealed and shaken, and placed into a Wallac Trilux luminescence counterfor cAMP detection. Effective 50% concentrations (EC50) were calculatedusing Origin software (OriginLab, Northampton, Mass.).

Acylated glucagon-based co-agonist peptides were prepared. In vitroresults for a selection of these peptides are shown in Table 13.Although the unacylated peptide, like native glucagon, was insoluble inphosphate-buffered saline solutions at 1 mg/mL concentrations, acylationwas observed to enhance solubility of the peptide at neutral pH.

TABLE 13 Receptor Activation Curves and nM EC₅₀ values for AcylatedPeptides GLP-1 Glucagon Peptide Receptor N Receptor N GLP-1 0.04 15 >1003 E16 K20-glucagon-NH2 0.21 9 0.18 10 E16 K20-glucagon-NH2 with K¹⁰-C₁₆0.09 8 0.40 8 E16 K20-glucagon-NH2 with 0.05 8 0.14 8 K¹⁰-W-C₁₆ E16K20-glucagon-NH2 with K¹⁰-C₁₈ 0.03 8 0.12 8 E16 K20-glucagon-NH2 with0.04 11 0.05 12 K¹⁰-W-C₁₈ Glucagon 7.42 6 0.07 17

All four acylated peptides exhibited increased potency at the GLP-1receptor. Inclusion of the tryptophan spacer provided better potency atthe glucagon receptor. An acyl chain length of C18 is slightlypreferred.

While acylation can extend the half-life of a peptide to hours or more,PEGylation with repeats in tens of kDa ranges can do even more. Peptidescomprising both types of modifications were prepared. These peptides areexpected to exhibit extended half-life in circulation, as well asresistance to DPP-IV and other proteases. In vitro results for aselection of these peptides are shown in Table 14.

TABLE 14 Receptor Activation Curves and nM EC₅₀ values for Acylated,PEGylated Peptides GLP-1 Glucagon Peptide Receptor N Receptor N GLP-10.04 15 >100 3 E16 K20-glucagon-NH2 0.21 9 0.18 10 (SEQ ID NO: 545) E16K20-glucagon-NH2 with K¹⁰-W- 0.23 13 0.52 13 C₁₆ and C²⁴-40K PEG (SEQ IDNO: 546) E16 K20-glucagon-NH2 with K¹⁰-C₁₈ 0.15 12 0.84 13 and C²⁴-40KPEG (SEQ ID NO: 547) E16 K20-glucagon-NH2 with K¹⁰-W- 1.64 3 1.30 5 C₁₈and C²⁴-40K PEG (SEQ ID NO: 548) Glucagon (SEQ ID NO: 1) 7.42 6 0.07 17

Two of the three peptides retained their high potency at both the GLP-1and glucagon receptors, with an EC50 of less than 1 nM. The K¹⁰—W—C₁₈acylated and PEGylated peptide exhibited about ten-fold potency lossesat both receptors. This series of peptides shows that the position tenacylation is compatible with a PEGylation in the C-terminal portion ofthe glucagon peptide, e.g. position 24, 28 or 29, within a C-terminalextension, or at the C-terminus (e.g., through adding a C-terminal Cys).

Example 20

Various acylated glucagon co-agonist peptides were made as essentiallydescribed in Example 19 and tested for in vivo activity. Specifically,Peptide A (SEQ ID NO:1 modified to contain AIB at position 2, Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Ile at position 23, Cys at position 24, which Cys isbonded to a 40K PEG, and C-terminal amide) was further modified tocomprise a Lys at position 10. The Lys10 was acylated with a C8 fattyacid chain, a C14 fatty acid chain, a C16 fatty acid chain, or a C18fatty acid chain.

Activity at the GLP-1 receptor of each of the acylated peptides wasassayed as described in Example 14 and compared to the activity of GLP-1(7-37)acid (SEQ ID NO: 50) as a control. The EC50 of each of theacylated peptides at the GLP-1 receptor shown in Table 15 is similar tothe EC50 of the GLP-1 peptide.

TABLE 15 GLP-1 Receptor Activation Potency EC₅₀ (nM) GLP-1 0.0222 ±0.0002 Peptide A K¹⁰-C₈ 0.0174 ± 0.0004 Peptide A K¹⁰-C₁₄ 0.0168 ±0.0004 Peptide A K¹⁰-C₁₆ 0.0127 ± 0.0003 Peptide A K¹⁰-C₁₈ 0.0118 ±0.0002

The peptides were then tested in vivo by subcutaneously injectingdiet-induced obesity (DIO) mice with various acylated and non-acylatedpeptides, or vehicle alone, QW (70 nmol/kg/week) for 2 weeks. 6 mice pergroup with initial average body weight of 44 g were tested. Body weight,body composition, food intake, and blood glucose levels were determinedperiodically.

As shown in FIG. 11, the acylated peptides are able to cause weight lossto a similar extent than the non-acylated peptide. As shown in FIG. 11,between about 7 and 12% weight loss is achieved within the first 3 daysof treatment with the acylated peptides. As shown in FIG. 12, theacylated peptides caused a decrease in food intake. Furthermore, asshown in FIG. 13, the ad libitum blood glucose levels of the acylatedpeptides were reduced after 1 day of treatment.

Example 21

The following acylated glucagon co-agonist peptides were made asessentially described in Example 19.

(A) “Chimera-2 Aib2 Lys10-C18 Cys24(40K)”: native glucagon amino acidsequence (SEQ ID NO: 1) comprising the following modifications: Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Ile at position 23, and Ala at position 24, and aC-terminal amide (“Chimera 2”), further modified with AIB at position 2,a Lys 10 acylated with a C18 fatty acid and a Cys at position 24pegylated with a 40K PEG group;(B) “Chimera-2 Aib2 Lys10-C16 Cys24(40K)”: Chimera 2 further modifiedwith AIB at position 2, a Lys10 acylated with a C16 fatty acid and aCys24 pegylated with a 40K PEG group;(C) “Glucagon Lys10-C18 E16 K20 Cys24(40K)”: native glucagon amino acidsequence (SEQ ID NO: 1) comprising the following modifications: Glu atposition 16, Lys at position 20, and C-terminal amide(“E16K20-glucagon-NH2”) was further modified with a Lys10 acylated witha C18 fatty acid and a Cys24 pegylated with a 40K PEG group;(D) “Glucagon Lys10-TrpC16 E16 K20 Cys24(40K)”: E16K20-glucagon-NH2 wasfurther modified with Lys10 linked to a Trp spacer which was acylatedwith a C16 fatty acid;(E) “Glucagon Lys10-TrpC18 E16 K20 Cys24(40K)”: E16K20-glucagon-NH2 wasfurther modified with Lys10 linked to a Trp spacer which was acylatedwith a C18 fatty acid.

The acylated glucagon co-agonist peptides were tested for theiractivities at the Glucagon and GLP-1 receptors generally as described inExample 14. The EC50 at each of the glucagon receptor and the GLP-1receptor in comparison to controls (GLP-1 (7-37) OH (amino acids 7-37 ofGLP-1), Glucagon (1-29)OH (SEQ ID NO: 1), and Chimera 2 Cys24 (40K)(Chimera 2 with a 40K PEG on Cys 24)) are as shown in Table 16.

TABLE 16 EC50 at EC50 at Glucagon GLP-1 Receptor Receptor (nM) (nM)GLP-1 (7-37) OH >1000.00 0.04 Glucagon (1-29) OH 0.07 7.5 Chimera 2Cys24 (40K) 2.83 0.04 Chimera-2 Aib2 Lys10-C18 Cys24 (40K) 8.55 0.14Chimera-2 Aib2 Lys10-C16 Cys24 (40K) 17.41 0.05 Glucagon Lys10-C18 E16K20 Cys24 (40K) 0.84 0.15 Glucagon Lys10-TrpC16 E16 K20 Cys24 (40K) 0.540.23 Glucagon Lys10-TrpC18 E16 K20 Cys24 (40K) 1.29 1.64

Example 22

The following acylated glucagon co-agonist peptides were made asessentially described in Example 19:

(A) Peptide A: native glucagon amino acid sequence (SEQ ID NO: 1)comprising the following modifications: Glu at position 16, Lys atposition 20, and C-terminal amide (“E16K20-glucagon-NH2”);(B) Peptide B: E16K20-glucagon-NH2 further comprising a Lys10 acylatedwith a C16 fatty acid;(C) Peptide C: E16K20-glucagon-NH2 further comprising a Lys10 acylatedwith a C18 fatty acid;(D) Peptide D: E16K20-glucagon-NH2 further comprising a Lys10 linked toa Glu (a spacer residue) acylated with a C16 fatty acid;(E) Peptide E: E16K20-glucagon-NH2 further comprising a Lys10 linked toa Trp (a spacer residue) acylated with a C18 fatty acid.

The activity of the peptides were assayed generally according to Example14 and the EC50 at each of the glucagon receptor and the GLP-1 receptorare shown in Table 17.

TABLE 17 EC₅₀ at EC₅₀ at Glucagon GLP-1 Receptor Receptor (nM) (nM)GLP-1 OH >1000 0.037 Glucagon (1-29) OH (SEQ ID NO: 1) 0.098 10 PeptideA 0.203 0.188 Peptide B 0.236 0.125 Peptide C 0.086 0.032 Peptide D0.062 0.056 Peptide E 0.044 0.031

Example 23

A glucagon co-agonist peptide was made comprising the amino acidsequence of SEQ ID NO: 1 with the following modifications: Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Ile at position 23, Ala at position 24, Val atposition 27, Lys at position 28 and C-terminal amide (“Chimera 1”).C-terminally truncated versions of Chimera 1 were made by deleting theamino acid at position 29 of Chimera 1 (“Chi 1 (1-28)”), or by deletingamino acids at both positions 28 and 29 of Chimera 1 (“Chi 1 (1-27)”).

A glucagon peptide comprising the amino acid sequence of SEQ ID NO: 1with the following modifications: Glu at position 16, C-terminal amide(“E16 Gluc-NH2”) was also C-terminally truncated, by deleting either theamino acid at position 29 (“E16 GlucNH2 (1-28)”) or by deleting aminoacids at both positions 28 and 29 (“E16 GlucNH2 (1-27)”).

The activity at the glucagon receptor and the GLP-1 receptor of thetruncated peptides, as well as the non-truncated peptides, were assayedfor functional activity generally according to Example 14. Deletion ofamino acids at positions 28 and 29 of the E16 GlucNH2 peptide or theChimera 1 peptide did not significantly impact the activity of thepeptide at the glucagon receptor. Deletion of amino acids at positions28 and 29 of E16 GlucNH2 did not appreciably change the potency of thepeptide at the GLP-1 receptor. Deletion of amino acids at positions 28and 29 of Chimera 1 did not impact its activity at the GLP-1 receptor.

Deletion of the amino acid at position 29 of either the Chimera 1peptide or the E16 GlucNH2 peptide did not significantly impact theactivity at either the glucagon receptor or the GLP-1 receptor.

Example 24

Diet-induced obesity (DIO) mice were injected intraperitoneally at the−15 min time point with 0.2, 2, 20, or 70 nmol/kg of one of thefollowing:

(A) vehicle only,

(B) native glucagon amino acid sequence (SEQ ID NO: 1) comprising thefollowing modifications: Glu at position 16, Gln at position 17, Ala atposition 18, Lys at position 20, Glu at position 21, Ile at position 23,and Ala at position 24, and a C-terminal amide (“Chimera 2”) furthermodified to comprise AIB at position 2 and Cys at position 24, which Cysis pegylated with a 40K PEG (“Chimera-2-AIB² Cys²⁴-40 kD”),

(C) Chimera 2 further modified to comprise AIB at position 2, Lys atposition 10, which Lys is acylated with a C8 fatty acid, and Cys atposition 24, which Cys is pegylated with a 40K PEG (“Chimera-2 AIB²K¹⁰—C8 Cys²⁴-40 kD”), or

(D) Chimera 2 further modified to comprise AIB at position 2, Lys atposition 10, which Lys is acylated with a C16 fatty acid, and Cys atposition 24, which Cys is pegylated with a 40K PEG (“Chimera-2 AIB²K¹⁰—C16 Cys²⁴-40 kD”).

A saline solution comprising 25% (v/v) glucose was injected at a dose of1.5 g/kg of body weight at the 0 min time point. Blood glucose levelswere measured at the −15, 0, 15, 30, 60, and 120 min time points.

FIGS. 15-17 show the blood glucose levels (mg/dL) of mice injected with2, 20, and 70 nmol/kg, respectively, at the indicated time points. Forall doses tested, Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40 kD demonstrated thegreatest ability to lower blood glucose in the mice. As shown in FIG.17, this peptide had similar activity as Chimera-2-AIB² Cys²⁴-40 kD.

Example 25

DIO mice were injected intraperitoneally at the −24 hr time point with70 nmol/kg of one of the following:

(A) vehicle only,

(B) Chimera-2-AIB² Cys²⁴-40 kD, as described above in Example 24,

(C) Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40 kD, as described above in Example 24,or

(D) Chimera-2 AIB² K¹⁰—C16 Cys²⁴-40 kD, as described above in Example24.

A saline solution comprising 25% (v/v) glucose was injected at a dose of1.5 g/kg of body weight at the 0 min time point. Blood glucose levelswere measured at the 0, 15, 30, 60, and 120 min time points.

FIG. 18 demonstrates the blood glucose levels (mg/dL) of the mice at theindicated time points. All three peptides demonstrate significantactivity at lowering blood glucose in the mice.

Example 26

DIO mice were injected intraperitoneally with vehicle only or 15 or 70nmol/kg of one of the following:

(A) Chimera-2-AIB² Cys²⁴-40 kD, as described above in Example 24,

(B) Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40 kD, as described above in Example 24,or

(C) Chimera-2 AIB² K¹⁰—C16 Cys²⁴-40 kD, as described above in Example24.

Body weight was measured before injection and at 1, 3, 5, and 7 dayspost-injection.

FIG. 19 demonstrates the % change of body weight for each group of mice.At both doses tested, Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40 kD andChimera-2-AIB² Cys²⁴-40 kD demonstrate comparable ability to lower bodyweight. At the higher dose tested, Chimera-2 AIB² K¹⁰—C16 Cys²⁴-40 kDdemonstrates significant ability to lower body weight

Example 27

A peptide of SEQ ID NO: 555, comprising a Tyrosine at position 1 and alactam bridge between E16 and K20, (and an amide in place of theC-terminal carboxylate) was synthesized as essentially described aboveand tested in vitro for activity at GLP-1 and glucagon receptors byExample 14. The EC50 of the peptide at each receptor is shown in Table18.

TABLE 18 Receptor EC₅₀ (nM) Std. Dev Relative Activity Glucagon 0.0440.151 343.18% GLP-1 0.062 0.062 100.00% Relative activity is activityrelative to the native hormone of the indicated receptor.

Based on these data, it was determined that the peptide of SEQ ID NOs:555 was an exemplary glucagon/GLP-1 co-agonist peptide.

Example 28

A peptide of SEQ ID NO: 1 (Glucagon(1-29)), a peptide of SEQ ID NO: 1with an amide replacing the C-terminal carboxylate (Glucagon (1-29a)),and a peptide of SEQ ID NO: 1 with AIB at each of positions 2 and 16 andan amide replacing the C-terminal carboxylate (Glucagon(1-29a) Aib²Aib¹⁶) were synthesized as essentially described above. These peptideswere then tested in vitro for activity at the GLP-1 receptor andglucagon receptors by the methods described in Example 14. The EC50 ofeach peptide are shown in Table 19.

TABLE 19 Glucagon Receptor GLP-1 Receptor Peptide EC₅₀ (nM) SD EC₅₀ (nM)SD Glucagon (1-29) 0.04 0.01 3.65 0.21 Glucagon (1-29a) Aib² 0.09 0.020.10 0.01 Aib¹⁶ Glucagon (1-29a) ND ND 0.50 0.05 GLP-1(1-31)OH ND ND0.03 0.00 SD = standard deviation

Example 29

The following peptides were synthesized as essentially described above:

(1) Glucagon(1-29), as described in Example 28,

(2) Glucagon(1-29a) Aib² Aib¹⁶ (as described in Example 28) with a Cysat position 24 and a Lys at position 10 covalently bonded to a Trpcomprising a C16 fatty acid (“Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶Cys²⁴”)

(3) Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶ Cys²⁴ in which the Cyscomprises a 40 kD PEG group (“Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶Cys²⁴-40 kD”),

(4) Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶ Cys²⁴ comprising Aib atposition 20 (“Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶ Aib²⁰ Cys²⁴),and

(5) Glucagon (1-29a) Aib² Lys¹⁰-Trp-C16 Aib¹⁶ Aib²⁰ Cys²⁴ in which theCys comprises a 40 kD PEG group (“Glucagon (1-29a) Aib²Lys¹⁰-Trp-C16Aib¹⁶ Aib²⁰ Cys²⁴-40 kD).

These peptides were then tested in vitro for activity at the GLP-1receptor and glucagon receptors by the methods of Example 14. The EC50of each peptide are shown in Table 20.

TABLE 20 Glucagon Receptor GLP-1 Receptor Peptide EC₅₀ (nM) SD EC₅₀ (nM)SD Glucagon (1-29) 0.04 0.01 Glucagon (1-29a) Aib² 0.25 0.02 0.24 0.03Lys¹⁰-Trp-C16 Aib¹⁶ Cys²⁴ Glucagon (1-29a) Aib² 0.29 0.03 0.19 0.02Lys¹⁰-Trp-C16 Aib¹⁶ Cys²⁴-40K Glucagon (1-29a) Aib² 2.06 0.02 1.15 0.19Lys¹⁰-Trp-C16 Aib¹⁶ Aib²⁰ Cys²⁴ Glucagon (1-29a) Aib² 2.37 0.24 0.600.06 Lys¹⁰-Trp-C16 Aib¹⁶ Aib²⁰ Cys²⁴-40K GLP-1(1-31)OH 0.02 0.01

Example 30

The in vivo effects of acylated and pegylated glucagon peptides weretested in DIO mice. Specifically, 6 groups of DIO mice (8 mice pergroup), each group having an average initial body weight of 58 g, wereinjected intraperitoneally with 10, 20, 40, or 80 nmol/kg of an acylatedand pegylated glucagon peptide or a vehicle control once a week for 2weeks. The acylated and pegylated glucagon peptides used in the studywere Chimera-2 AIB² K¹⁰—C8 Cys²⁴-40 kD (as described in Example 26) andPeptide A K¹⁰—C₁₄ (as described in Example 20).

Changes in body weight of and food intake by the mice were measured 0,1, 3, 5, 7, 8, 10, 12, and 14 days after injection. Blood glucose levelsof the mice were monitored throughout the 14 days. Glucose tolerancetests were performed by injecting a 25% glucose in saline solution 1hour or 24 hours after administration of the acylated or pegylatedpeptide and measuring blood glucose levels at −60, 0, 15, 30, 60, or 120min after the glucose injection.

As shown in FIG. 20, the total body weight of mice injected with 40 or80 nmol/kg of acylated and pegylated Peptide A K¹⁰—C₁₄ was reduced ascompared to mice injected with the vehicle control.

As shown in FIG. 21, the blood glucose levels of mice injected with 20,40, or 80 nmol/kg Peptide A K¹⁰—C₁₄ or with 20 nmol/kg Chimera-2 AIB²K¹⁰—C8 Cys²⁴-40 kD in response to a glucose injection are lowered incomparison to vehicle control.

Example 31

Acylated glucagon analog peptides comprising or lacking a covalentintramolecular bridge were made by solid-phase synthesis and tested forin vitro activity at the glucagon and GLP-1 receptors. The EC50 (nM) ateach receptor and the % activity of the peptide relative to the nativepeptide at the corresponding receptor is shown in Table 21.

TABLE 21 EC₅₀ at % EC₅₀ at the % the GLP-1 Activity Glucagon ActivitySEQ receptor of receptor of Peptide Name ID NO: (nM) GLP-1 (nM) GlucagonDMIA1, 607 0.050   30% 0.027 203.7%  K10(C14), [E16/K20]- Gluc AmideDMIA1, 608 0.015  100% 0.014  392% K10(C16), [E16/K20]- Gluc AmideDMIA1, 609 0.011  136% 0.13 42.3% K10(C18), [E16/K20]- Gluc Amide AIB2,AIB16, 610 0.024 33.3% 0.044 77.3% K10(C14) Gluc Amide AIB2, AIB16, 6110.011 72.3% 0.020  170% K10(C16) Gluc Amide AIB2, AIB16, 612 0.009 88.9%0.016 212.5%  K10(C18) Gluc Amide dS2, E16/K20, 613 0.128  6.3% 0.15521.9% K10(C14) Gluc Amide dS2, E16/K20, 614 0.041 19.5% 0.076 44.7%K10(C16) Gluc Amide dS2, E16/K20, 615 0.025   60% 0.028  196% K10(C18)Gluc Amide

Several glucagon analogs lacking a covalent intramolecular bridge andcomprising an AIB at position 2, an AIB at position 16, and a fatty acylgroup attached via a spacer to a Lys residue at position 10 were made asessentially described herein. The acylated glucagon analogs differed bythe type of spacer, the presence or absence of pegylation, and/or by thesize of the acyl group. The acylated glucagon analogs were tested for invitro activity at the glucagon receptor and the GLP-1 receptor asessentially described in Example 14. A summary of the structure and invitro activity at the glucagon and GLP-1 receptors of each peptide isshown in Tables 22 and 23.

TABLE 22 Glucagon analog backbone amino acid sequence:HXQGTFTSDKSKYLDXRRAQDFVQWLMNT-NH₂ wherein X = AIB (SEQ ID NO: 562) Sizeof EC₅₀ at SEQ Fatty Glucagon EC₅₀ at GLP-1 Peptide ID Acyl ReceptorReceptor Name NO: Spacer Group (nM) (nM) wt 1 n/a n/a 0.031 ± 0.014glucagon wt GLP-1 n/a n/a 0.036 ± 0.010 26 637 None None 0.653 ± 0.2850.475 ± 0.046 50 563 None C16 0.572 ± 0.084 0.291 ± 0.060 82 564 Ala-AlaC16 0.024 ± 0.001 0.108 ± 0.018 83 565 γ-Glu-γ- C16 0.014 ± 0.002 0.043± 0.005 Glu 84 566 β-Ala-β- C16 0.011 0.004 Ala 85 567 6-amino- C160.010 0.005 hexanoic acid 86 568 Leu-Leu C16 0.011 0.006 87 569 Pro-ProC16 0.017 0.009  77* 570 None C14 21.94 ± 14.47 1.458 ± 0.132  78* 571γ-Glu-γ- C14 0.319 ± 0.091 0.103 ± 0.023 Glu  81* 573 Ala-Ala C14 0.597± 0.175 0.271 ± 0.019  79* 575 Ala-Ala C16 0.102 ± 0.011 0.055 ± 0.001 80* 576 γ-Glu-γ- C16 0.108 ± 0.028 0.042 ± 0.008 Glu *indicates thatthe peptide comprised a Cys residue at position 24 (in place of Gln)which Cys was covalently attached to a 40 kDa PEG group.

TABLE 23 Glucagon analog backbone amino acid sequence:HXQGTFTSDKSKYLDXRRAQDFVWLMNT-NH₂ wherein X = AIB (SEQ ID NO: 562) Sizeof EC₅₀ at EC₅₀ at Fatty Glucagon GLP-1 Peptide SEQ ID Acyl ReceptorReceptor Name NO: Spacer Group (nM) (nM) wt 1 n/a n/a 0.008 ± 0.003glucagon wt GLP-1 n/a n/a 0.004 ± 0.001 77** 616 none C14 0.144 ± 0.0290.063 ± 0.012 78** 617 γ-Glu-γ- C14 0.009 ± 0.001 0.008 ± 0.001 Glu 81**618 Ala-Ala C14 0.027 ± 0.006 0.018 ± 0.001 80** 619 γ-Glu-γ- C16 0.006± 0.001 0.008 ± 0.001 Glu 79** 620 Ala-Ala C16 0.010 ± 0.001 0.008 ±0.001 **peptide comprising Cys at position 24 (in place of Gln) whichCys was not covalently attached to a PEG molecule

As shown in Tables 22 and 23, the peptides comprising a fatty acyl groupattached via a spacer significantly increased their potency as comparedto peptides comprising a fatty acyl group attached directly to thepeptide backbone.

Example 32

DIO mice (8 mice per group), each with an average bodyweight of 48.7 g,were subcutaneously injected daily for seven days with vehicle only,with 30 nmol/kg or 100 nmol/kg of an acylated glucagon analog peptide,or with the long-acting GLP-1 analog, Liraglutide (Novo Nordisk,Denmark). The acylated glucagon analogs were as follows:

“(C16) Glucagon Amide” comprised the amino acid sequence of wild-typeglucagon (SEQ ID NO: 1) with the Tyr at position 10 modified to anacylated Lys residue, wherein the acylated Lys comprised a C16 fattyacyl group, and the C-terminal carboxylate replaced with an amide group;

“γE-γE-C16 Glucagon Amide” comprised the same structure of C16 GlucagonAmide, except that the C16 fatty acyl group was attached to the Lys atposition 10 through a gamma-Glu-gamma-Glu dipeptide spacer (seestructure of acylated Lys below);

“AA-C16 Glucagon Amide” comprised the same structure of C16 GlucagonAmide, except that the C16 fatty acyl group was attached to the Lys atposition 10 through an Ala-Ala dipeptide spacer; and

“βAβA-C16 Glucagon Amide” comprised the same structure of C16 GlucagonAmide, except that the C16 fatty acyl group was attached to the Lys atposition 10 through an β-Ala-β-Ala dipeptide spacer.

The body weight of the mice was monitored daily and the total change inbody weight (%) is shown in FIG. 22. As shown in FIG. 22, most of theacylated glucagon peptides at each dose caused a reduction in bodyweight. While Liraglutide demonstrated an approximate 12% decrease inbody weight, the glucagon analog peptide γE-γE-C16 Glucagon Amideexhibited the greatest ability to cause weight loss in mice at thematched dose. Even the lower dose of γE-γE-C16 Glucagon Amide caused asubstantial decrease in body weight.

The fat mass of the mice was measured on Day 7 of the study. As shown inFIG. 23, the mice which were administered 100 nmol/kg γE-γE-C16 GlucagonAmide exhibited the lowest fat mass.

Blood glucose levels of the mice were also monitored during the courseof the assay. As shown in FIG. 24, the glucagon analog peptide γE-γE-C16Glucagon Amide at the higher dose worked as well as Liraglutide todecrease blood glucose levels in mice.

Example 33

Acylation of a glucagon analog peptide having GLP-1 activity wasevaluated as follows. A non-acylated glucagon analog peptide comprisingthe structure of Chimera 2 with AIB at position 2 and Cys at position 24(comprising a 40 kDa PEG molecule) was modified to comprise an acylatedLys residue at position 10. The non-acylated glucagon analog peptidecomprised the amino acid sequence of SEQ ID NO: 580. The Lys at position10 was acylated with a C8, C14, C16, or C18 fatty acyl group and theacylated peptides comprised the structures of SEQ ID NOs: 534-537,respectively. The in vitro activity at the GLP-1 receptor of thenon-acylated peptide and acylated versions thereof were tested asessentially described herein. The EC50 at the GLP-1 receptor of eachpeptide is shown in Table 24.

TABLE 24 Glucagon analog peptide sequenceHXQGTFTSDYSKYLDEQAAKEFICWLMNT-NH₂, wherein X = AIB (SEQ ID NO: 580) EC₅₀(nM) SD GLP-1 0.026 0.003 Non-acylated Glucagon analog peptide (SEQ IDNO: 580) 0.095 0.015 C₈ acylated Glucagon analog peptide (SEQ ID NO:534) 0.058 0.002 C₁₄ acylated Glucagon analog peptide (SEQ ID NO: 535)0.044 0.005 C₁₆ acylated Glucagon analog peptide (SEQ ID NO: 536) 0.0330.005 C₁₈ acylated Glucagon analog peptide (SEQ ID NO: 537) 0.011 0.001

Example 34

Glucagon analog peptides were made by solid-phase peptide synthesis asdescribed herein and were acylated at either position 10 or 30 of thepeptide. The peptides and their structure were as follows:

“Peptide dS2E16K20K30-C14 Gluc Amide” comprised the amino acid sequenceHXQGTFTSDYSKYLDERRAKDFVQWLMNTK-amide (SEQ ID NO: 581), wherein the X atposition 2 is d-Ser, wherein the Lys at position 30 is acylated with aC14 fatty acyl group, and the C-terminal carboxylate is replaced with anamide;

“Peptide dS2K10(C14)E16K20-Gluc Amide” comprised the amino acid sequenceHXQGTFTSDKSKYLDERRAKDFVQWLMNT-amide (SEQ ID NO: 582); wherein the X atposition 2 is d-Ser, wherein the Lys at position 10 is acylated with aC14 fatty acyl group, and the C-terminal carboxylate is replaced with anamide;

“Peptide dS2E16K20K30-C16 Gluc Amide” comprised the amino acid sequenceHXQGTFTSDYSKYLDERRAKDFVQWLMNTK-amide (SEQ ID NO: 583), wherein the X atposition 2 is d-Ser, wherein the Lys at position 30 is acylated with aC16 fatty acyl group, and the C-terminal carboxylate is replaced with anamide;

“Peptide dS2K10(C16)E16K20-Gluc Amide” comprised the amino acid sequenceHXQGTFTSDKSKYLDERRAKDFVQWLMNT-amide (SEQ ID NO: 584); wherein the X atposition 2 is d-Ser, wherein the Lys at position 10 is acylated with aC16 fatty acyl group, and the C-terminal carboxylate is replaced with anamide;

“Peptide Chimera 2-AIB2-K10-acylated” comprised the amino acid sequenceHXQGTFTSDKSKYLDEQAAKEFICWLMNT-amide (SEQ ID NO: 585); wherein the X atposition 2 is AIB, the K at position 10 is acylated with a C18 fattyacyl group, Cys at position 24 comprises a 40 kDa PEG molecule, and theC-terminal carboxylate is replaced with an amide; and

“Peptide Chimera 2-AIB2-K30-acylated” comprised the amino acid sequenceHXQGTFTSDYSKYLDEQAAKEFICWLMNTK-amide (SEQ ID NO: 586), wherein the X atposition 2 is AIB, the K at position 10 is acylated with a C18 fattyacyl group, Cys at position 24 comprises a 40 kDa PEG molecule, and theC-terminal carboxylate is replaced with an amide.

The in vitro activity at the GLP-1 receptor and glucagon receptor ofeach peptide was tested as essentially described in Example 14. Theresults are shown in Table 25.

TABLE 25 Position EC50 at EC50 at at which the the acyl glucagon GLP-1group is receptor receptor Peptide Name found (nM) (nM) PeptidedS2E16K20K30-C14 Gluc 30 3.53 0.84 Amide Peptide dS2K10(C14)E16K20-Gluc10 0.155 0.041 Amide Peptide dS2E16K20K30-C16 Gluc 30 4.89 3.05 AmidedS2K10(C16)E16K20-Gluc Amide 10 0.076 0.041 Peptide Chimera2-AIB2-K10-acylated 30 N/A 0.465 Peptide Chimera 2-AIB2-K30-acylated 10N/A 0.007

Example 35

Solid-phase peptide synthesis was employed for the assembly of thesequence, XSQGTFTSDYSKYLDERRAKDFVCWLMNT-NH₂, wherein X=DMIA (SEQ ID NO:587). After selective deprotection of the Glu at position 16 and the Lysat position 20, the peptide was cyclized via a lactam bridge on resin.The crude peptide after cleavage was then purified by preparativeRP-HPLC and characterized by MS (calc. for [M+H]: 3479.9. found 3480.9).PEGylation was conducted by mixing the peptide precursor andiodoacetyl-functioned 40 k Da PEG (NOF)(1:1) in 7 M urea/50 mM Trisbuffer, pH 8.5, at room temperature for 45 minutes to form a covalent,thioether bond between the PEG and a Cys of the peptide, as shown below

The PEGylated peptide was purified by preparative HPLC and the desiredfractions were collected and lyophilized to yield a off-white powder.The product was confirmed by MALDI-TOF-MS (44000-46000, broad peak).

The in vitro activity at the GLP-1 receptor and glucagon receptor weretested as essentially described in Example 14. The EC50s at the GLP-1receptor and glucagon receptor were 0.327 nM and 0.042 nM, respectively.

Example 36

Solid-phase peptide synthesis was employed for the preparation of thepeptide precursor, HXEGTFTSDYSKYLDEQAAKEFICWLMNT-NH₂, wherein X=AIB (SEQID NO: 589). The crude peptide was then purified by preparative RP-HPLCand characterized by MS (calc. for [M+H]: 3412.8. found 3413.9).PEGylation was conducted by mixing the peptide precursor andiodoacetyl-functioned 40 k Da PEG (NOF)(1:1) in 7 M urea/50 mM Trisbuffer, pH 8.5, at room temperature for 45 minutes to form a covalent,thioether bond between the PEG and a Cys of the peptide, as shown below

The PEGylated peptide was purified by preparative HPLC and the desiredfractions were collected and lyophilized to yield a off-white powder.The product was confirmed by MALDI-TOF-MS (44000-46000, broad peak).

The in vitro activity at the GLP-1 receptor and glucagon receptor weretested as essentially described in Example 14. The EC50s at the GLP-1receptor and glucagon receptor were 0.027 nM and 33 nM, respectively.

Example 37

The following glucagon analog peptides comprising a backbone of PeptideJ

(SEQ ID NO: 591) HS-X-GTFTSDYSKYLDTRRAAEFVAWL(Nle)DE

or Peptide K

(SEQ ID NO: 592) HS-X-GTFTSDYSKYLD(Aib)RRAADFVAWLMDEwith additional modification at position 3 were made by solid-phasepeptide synthesis as essentially described herein. The peptides weretested for in vitro activity at the glucagon receptor as essentiallydescribed in Example 14. The EC50 (nM) of each peptide is shown in Table26.

TABLE 26 EC50 at Peptide Amino Acid Glucagon Backbone at Position 3 SEQID NO: Receptor (nM) % activity* J Q 593 0.24 25% J C(Acm) 594 0.18 33%J Dab(Ac) 595 0.31 19% J Dap(urea) 596 0.48 13% J Q(Me) 597 0.48 13% JM(O) 598 0.91 7% J Orn(Ac) 599 0.92 7% K Q 600 0.39 15% K Dab(Ac) 6010.07 86% K Q(Me) 602 0.11 55% Q = glutamine; C(Acm) =acetamidomethyl-cysteine; Dab(Ac) = acetyldiaminobutanoic acid;Dap(urea) = carbamoyldiaminopropanoic acid; Q(Me) = methylglutamine;M(O) = methionine-sulfoxide; Orn(Ac) = acetylornithine.

As shown in Table 26, multiple amino acids could be placed at position 3without a substantial loss of activity at the glucagon receptor, and, insome cases, the modification actually increased the activity, e.g.,Dab(Ac) and Q(Me) on the Peptide K backbone.

Example 38

Glucagon analog peptides comprising Dab(Ac) at position 3 on variousglucagon analog backbones were made as essentially described herein andthe in vitro activity at the glucagon receptor was tested. Thestructures and activities of each peptide are shown in Table 27.

TABLE 27 EC50 SEQ (nm) at ID Glucagon % Amino acid sequence NO: Receptoractivity Wildtype Glucagon 1 0.026 100 HSQGTFTSDYSKYLDSRRAQDFVQWLMDT 6420.015 173 HSDab(Ac)GTFTSDYSKYLDAibRRAADFVAWLLDE 603 0.069 37HSDab(Ac)GTFTSDYSKYLDAibRRAADFVAWLLDTGPSSGAPP 604 0.023 113 PS amideHSDab(Ac)GTFTSDYSKYLDAibRRASDFVSWLLDE 605 0.048 54HSDab(Ac)GTFTSDYSKYLDAibRRATDFVTWLLDE 606 0.057 46

Example 39

A first glucagon analog peptide (AIB2, AIB16, K10(C16) Gluc Amide)comprising SEQ ID NO: 1 with AIB at positions 2 and 16, Lys at position10, wherein the Lys at position 10 was covalently attached to a C16fatty acyl group, and an amide in place of the C-terminal carboxylatewas made as essentially described herein. A second glucagon analogpeptide (AIB2, AIB16, K10(C16), K30 Gluc Amide) having the samestructure as the first glucagon analog peptide, except that a Lys wasadded to the C-terminus. The in vitro activity of the peptides wastested as essentially described in Example 14 and was additionallytested in a solution comprising 20% human plasma. The EC50 (nM) at eachreceptor for the peptides is shown in Table 28.

TABLE 28 EC50 (nm) at EC50 (nm) Glucagon at GLP-1 EC50 (nm) ReceptorReceptor at (20% EC50 (nm) (20% SEQ Glucagon human at GLP-1 humanAmino Acid Sequence ID NO: Receptor plasma) Receptor plasma) Glucagon 10.026 0.046 GLP-1 0.022 0.028 AIB2, AIB16, K10(C16) Glucagon Amide 5630.052 0.023 0.026 0.014 AIB2, AIB16, K10(C16), K30 Glucagon Amide 6220.761 0.313 0.031 0.017

Example 40

The discovery of leptin documented the existence of an endocrine systemthat regulates energy balance and body adiposity. It also recruitedinterest and investment in obesity research as a means to identifyenvironmental and pharmacologic approaches to manage what has become aglobal epidemic of disease. Sufficiently efficacious and safepharmacologic treatment for obesity has yet to emerge and surgeryconstitutes the only proven option to sustained weight loss. It isreported herein that the combinatorial efficacy of receptor agonism attwo endocrine hormonal receptors to achieve potent satiety inducing andlipolytic effects in a single peptide of sustained duration of action.Two specific glucagon analogs with activity at the GLP1-R comparable tonative GLP-1, but differing from each other in their level of glucagonreceptor agonism were studied pharmacologically in rodent obesitymodels. Once weekly administration of these pegylated peptides selectedfrom a series of high potency analogs with differential glucagon andGLP-1 activity normalized adiposity and glucose tolerance levels in dietinduced obese mice (average body weight ca. 50 g) within a month. Bodyweight loss was a consequence of body fat loss resulting from decreasedfood intake and increased energy expenditure, which increased with thelevel of glucagon receptor agonism. These co-agonist compounds alsonormalized glucose and lipid metabolism including liver steatosis.Effects were dose dependent and successfully repeated in diet inducedobese rats. These preclinical studies indicate that when full GLP-1agonism is enhanced with an appropriate degree of glucagon receptoractivation, body fat reduction can be substantially and safelyaccelerated. The findings shown herein establish a basis for clinicaltesting and suggest an attractive novel treatment option for themetabolic syndrome.

Example 41

The following materials and methods pertain to the experiments describedin Examples 42 to 51.

Boc Peptide Synthesis and Cleavage.

Peptide syntheses were performed using 0.2 mmol 4-methylbenzhydrylamine(MBHA) resin (Midwest Biotech, Fishers, Ind.) on a modified AppliedBiosystems 430A peptide synthesizer. Solid-phase peptide synthesesutilized in situ neutralization for Boc-chemistry (Schnolzer, M. et al.,International Journal of Peptide Research and Therapeutics, 13:31-44(2007)). Completed peptidyl-resins were treated with HF/p-cresol (10:0.5v/v) at 0° C. for 1 h. HF was removed in vacuo and the deprotectedpeptide was precipitated and washed in diethyl ether. The peptide wasdissolved in 20% acetonitrile/1% acetic acid and lyophilized. Mostpeptides were prepared by Boc chemistry. The following side chainprotecting groups were used for Boc-amino acids (Midwest Biotech):Arg(Tos), Asp(OcHex), Asn(Xan), Glu(OcHex), His(BOM), Lys(2-Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CH0), Tyr(Br—Z). Peptide molecular weights wereconfirmed by electrospray ionization or MALDI-TOF mass spectrometry andpurified as described elsewhere.

Lactam Synthesis.

Cyclized peptides with i to i+4 lactam formation were synthesized onresin. Glu(OFm)-OH gamma ester (Peptides International, Louisville, Ky.)and Lys(Fmoc)-OH (Peptides International) were substituted forGlu(OcHex) and Lys(2-Cl—Z) at positions involved in lactam formation.The fully protected peptidyl-resin was treated with 20% piperidine inDMF for 45 minutes to remove Fmoc and OFm protecting groups. On resin,lactam formation was achieved after treatment with 5 equivalents ofbenzotriazole-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) (Fluka) in DMF/DIEA for 5 h. Lactam formation was confirmed byninhydrin analysis and mass reduction of 18 relative to the open form ofthe peptide.

Peptide Purification.

Following cleavage from the resin, crude peptide extracts were analyzedby analytical reverse-phase HPLC. Analytical separations were conductedin 0.1% TFA with an acetonitrile gradient on a Zorbax C8 column (0.46×5cm). After analytical analysis, the crude extract was purified bysemi-preparative chromatography in 0.1% TFA with an acetonitrilegradient on a Vydac C4 or C18 column (2.2×25 cm). Pegylated peptideswere purifed using the same conditions. Preparative fractions wereanalyzed for purity (>95%) by analytical reverse-phase HPLC utilizingthe conditions listed for analytical separations. Peptide masses andpurity were confirmed by electrospray ionization mass spectrometry(ESI-MS) or matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry. Pegylated peptides showed a broad massrange spanning 43400 by MALDI-TOF. Purified peptides were lyophilizedand stored at 4° C.

Pegylation of Peptides.

Purified peptides were mixed at a 1:1 molar ratio with methoxypoly(ethylene glycol) maleimido-propionamide-40K (Chirotech TechnologyLtd, Cambridge) in 7M urea/50 mM Tris, pH 8.0. Reaction progress wasmonitored by analytical reverse-phase HPLC and free peptide was consumedwithin 30 minutes. The reaction was quenched in 0.1% TFA, purified andcharacterized as described elsewhere.

Glucagon and GLP-I Receptor-Mediated cAMP Synthesis.

Each peptide analog was tested for its ability to stimulate cAMPproduction through the glucagon (Gcg) and GLP-1 receptors. HEK293 cellswere co-transfected with the GcgR or GLP-1R cDNAs and a luciferasereporter gene-linked to a cAMP response element (CRE). Cells were serumdeprived for 16 h by culturing in DMEM (Invitrogen, Carlsbad, Calif.)and supplemented with 0.25% Bovine Growth Serum (HyClone, Logan, Utah).Serial dilutions of Glucagon and GLP-1 analogs were added to 96-wellpoly-D-Lysine-coated plates (BD Biosciences, San Jose, Calif.)containing co-transfected HEK293 cells, and plates were incubated for 5h at 37° C., 5% CO₂. Following incubation, an equivalent volume (100 μL)of LucLite luminescence substrate reagent (Perkin-Elmer, Wellesley,Mass.) was added to each well and the plate was shaken for 3 min at 800rpm. The plate was incubated for 10 min in the dark and light output wasquantified on a MicroBeta1450 liquid scintillation counter(Perkin-Elmer, Wellesley, Mass.). Effective 50% concentrations (EC50)were calculated by Origin software (OriginLab, Northampton, Mass.).

Circular Dichroism Measurements.

Peptides were dissolved in 10 mM phosphate buffer pH 5.9 with increasingconcentrations of TFE, and peptide concentrations were quantified. Eachsample was diluted to 10 μM for CD measurements. CD data were collectedon a JASCO J-715 circular dichroism spectropolarimeter with constantnitrogen stream and temperature control of the 1 mm path length cell setat 25° C. Spectral data were accumulated for 5 scans from 270-190 nmwith a scan speed of 100 nm/min and 1 nm wavelength step. Solvent signalwas subtracted and data were smoothed (Savitzky and Golay, Anal. Chem.36:1627 (1964)); in the JASCO Spectra Manager software. Millidegreevalues obtained were converted to mean residue ellipticity with units ofdeg cm²dmol⁻¹. Calculated mean residue ellipticity values were inputinto DICHROWEB (Whitmore and Wallace, Biopolymers 89:392-400 (2008);Whitmore and Wallace, Nucleic Acids Research 32:W668-W673 (2004) toobtain percent helicity values.

Animals.

C57BI/6 mice were obtained from Jackson Laboratories and fed adiabetogenic diet from Research Diets, a high sucrose diet with 58% kcalfrom fat. Mice were single or group-housed on a 12:12-h light-dark cycleat 22° C. with free access to food and water. All studies were approvedby and performed according to the guidelines of the Institutional AnimalCare and Use Committee of the University of Cincinnati.

Body Composition Measurements.

Whole body composition (fat and lean mass) was measured using NMRtechnology (EchoMRI, Houston, Tex.).

Energy Balance Physiology Measurements.

Energy intake and expenditure, as well as home-cage activity, wereassessed by using a combined indirect calorimetry system (TSE Systems,Bad Homburg, Germany). Oxygen consumption and CO₂ production weremeasured every 45 min for a total of 120 h (including 12 h ofadaptation) to determine the respiratory quotient and energyexpenditure. Food and water intake and meal patterns were determinedcontinuously for 120 h at the same time as the indirect calorimetryassessments by integration of scales into the sealed cage environment.Meals were defined as food intake events with a minimum duration of 60s, and a break of 300 s between food intake events. Home-cage locomotoractivity was determined using a multidimensional infrared light beamsystem with beams scanning the bottom and top levels of the cage, andactivity being expressed as beam breaks. Stationary motor activity(fidgeting) was defined as consecutive breaks of one single light beamat cage-bottom level, ambulatory movement as breaks of any two differentlight beams at cage-bottom level, and rearing as simultaneous breaks oflight beams on both cage-bottom and the top level.

Blood Parameters.

Blood was collected after a 6-h fast from tail veins using EDTA-coatedMicrovette tubes (Sarstedt, Nuremberg, Germany) and immediately chilledon ice. After 15 min of centrifugation at 3,000 g and 4° C., plasma wasstored at −80° C. Plasma insulin was quantified by a radioimmunoassayfrom Linco (Sensitive Rat Insulin RIA; Linco Research, St. Charles,Mo.). Plasma TGs and cholesterol levels were measured by enzymatic assaykits (Thermo Electron, Waltham, Mass.). Samples were analyzedindividually with the exception that pooled samples (0.25 ml) from 5animals/group were subjected to fast-performance liquid chromatography(FPLC) gel filtration on two Superose 6 columns connected in series forlipoprotein separation. All assays were performed according to themanufacturer's instructions.

Glucose Tolerance Test.

For the determination of glucose tolerance, mice were subjected to 6 hof fasting and injected intraperitoneally (i.p.) with 2 g glucose/kgbody wt (50% D-glucose (Sigma) in 0.9% saline) for the glucose tolerancetest (GTT). Tail blood glucose levels (mg/dl) were measured by using ahand-held glucometer (TheraSense Freestyle) before (0 min) and at 15,30, 60, 90, and 120 min after injection.

Western Blot of WAT HSl.

Adipose tissue was placed in a 1.5-ml microfuge tube and lysed in icecold RIPA buffer (1×PBS, 1% Nonidet P40, 0.5% sodium doxycholate, 0.1%SDS with 50 mM NaF, 0.5 M phenylmethylsulfonyl fluoride, 0.1 mM NaVanadate, 20 ng/ml Aprotinin, 10 ng/ml Leupeptin) using a tissue lyser(Retsch, Inc Newtown, Pa. Cat. #85210) at 30 hz for 3 min. Samples werespun at 12,000 rpm for 15 min (4° C.) at which time the internatant wasremoved to a new tube and sonicated for 15 sec on ice. Samples were spunat 14,000 rpm for 10 min (4° C.) and the internatant was collected to anew tube. Samples were again spun at 19,000 rpm for 10 min (4° C.) andthe internatant collected to a new tube. An aliquot of sample was thentaken for protein assay. Samples were then boiled in 4×SDS/DTT bufferfor 2 min. 50 ng of protein from cell lysate were subjected to SDS/PAGEon 9% (w/v) acrylamide resolving gels and transferred to Hybond ECLnictrocellulose membranes. Membranes were blocked and probed withprimary antibodies of interest (HSL (4107)) from Cell Signaling;Phospho-HSL (ser 660) (4126) from Cell Signaling). After washing,primary antibody detection was performed using either HRP-conjugatedanti-(rabbit IgG) or anti-(mouse IgG) (HRP-conjugated anti-rabbit andanti-mouse secondary antibodies were purchased from Bio-Rad (170-6515 &170-6516)) and detected using enhanced chemiluminescence (AmershamBiosciences) and exposed to CL-Xposure film (Pierce).

Immunohistochemistry.

Paraffin embedded sections of white epididymal adipose tissue (5 μm)were stained with hematoxylin/eosin as described (Ogden, C. L. et al.JAMA 295:1549-1555 (2006)). For each individual mouse tissue block,adipocyte size of 100 cells from each of three different high-powerfields was quantified as areal measurement using Image Pro Plus 5.1software (Media Cybernetics, Bethesda, Md., USA).

Oil Red Staining.

To visualize lipid accumulation in liver tissue, 4-8 mm cross-sectionsof the livers that were harvested at sacrifice were stained with Oil Red0 dye. Images at both 20× and 40× magnification were acquired using a[compound-lens] microscope.

Quantitative RT-PCR Procedure.

Animals were sacrificed by decapitation in the fed state (1-4 h afterthe morning feeding) and various tissues were sampled, freeze-clamped,and stored at −80° C. for subsequent measurement of mRNA expression ofPEPCK, G6P, and HPRT (housekeeping) by real-time quantitative PCR(icycler, BioRad).

Total RNA was extracted from frozen tissue samples using a RNeasy LipidTissue Kit (Qiagen, Ca#74804) using the standard protocol. RNAconcentrations and purity were determined by spectrophotometry using theNanodrop. cDNA templates for RT-PCR were obtained using 2 μg of totalRNA. Reverse transcription reaction was performed with 10× DNase IReaction Buffer, DNase I, Amp Grade, 1 U/μl, depc-H₂O, 25 mM EDTA, 10 mMdNTP Mix, oligo(dT)₂₋₀ (50 μM), 5× First-Strand Buffer, 0.1 M DTT,RNaseOUT, and SuperScript 111 (Invitrogen).

The synthesized cDNAs were further amplified by PCR using thefluorescent dye SYBR green (BioRad, Ca#1708882) containing a finalconcentration of 0.5 μM of forward and reverse primers. Product puritywas confirmed by dissociation curves. No-template controls were includedin all assays, yielding no consistent amplification. A standard curvewas used to obtain the relative concentration of PEPCK or G6P, and theresults were corrected according to the concentration of HPRT, used ashousekeeping genes. The results are expressed as percent of vehicle,setting the mean of the vehicle group at 100% and then calculating eachindividual value of the 3 groups of animals studied.

Primer Sequences.

Primer sequences for PEPCK, G6P, and HPRT were taken from the NIHwebsite and primers were generated by IDT DNA.

Reverse Transcription and Quantitative Real-Time RT-PCR.

CD68 mRNA expression was quantified by real-time RT-PCR as described(Nomiyama, T. et al. Journal of Clinical Investigation 117:2877-2888(2007)). Briefly, upon sacrifice, 100 mg epididymal adipose tissue washomogenized in TRIZOL and total mRNA was reverse transcribed into cDNA.PCR reactions were performed using an iCycler (Bio-Rad) and SYBR Green Isystem (Bio-Rad). Each sample was analyzed in triplicate and normalizedto values for TFIIB mRNA expression. Mouse primer sequences used were asfollows:

CD68, (SEQ ID NO: 638) 5′-CAAGGTCCAGGGAGGTTGTG-3′ (forward),(SEQ ID NO: 639) 5′-CCAAAGGTAAGCTGTCCATAAGGA-3′ (reverse); and TFIIB,(SEQ ID NO: 640) 5′-CTCTCCCAAGAGTCACATGTCC, (SEQ ID NO: 641)5′-CAATAACTCGGTCCCCTACAAC-3′ (reverse).

Statistical Analyses.

Unless indicated otherwise, all statistical analyses were performedusing GraphPad Prism one-way ANOVAs and column statistics. Stated Pvalues are for one-way analysis of variance. All results are presentedas means±SE. (Receptor activation data is ±S.D.).

Example 42

Two glucagon peptides, Peptides X and Y, comprising the amino acidsequence of SEQ ID NO: 1 with amino acid modifications were made asdescribed herein. Both peptides comprised AIB at position 2, Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Ile at position 23, and Cys at position 24.Site-specific 40-kd pegylation was achieved at Cys at position 24through reaction with a maleimide-functionalized linear peg to yieldPeptide X-PEG and Y-PEG. Peptides Y and Y-PEG differed from Peptides Xand X-PEG, respectively, in that a single side-chain lactam bridge wasintroduced in the middle of Peptide Y or Peptide Y-PEG to stabilize thesecondary structure and enhance glucagon agonism. The two side chains ofGlu at position 16 and Lys at position 20 were covalently coupled in thecourse of peptide assembly as a side-chain amide. This macrocyclizationof the peptide represents a 21-atom lactam. Peptides X-PEG and Y-PEGwere tested for solubility and were found to be soluble in physiologicalbuffers at concentrations that exceed 25 mg/ml, and Peptides X-PEG andY-PEG proved completely resistant to ex vivo incubation with plasma forperiods of one week.

Example 43

The secondary conformation of peptides when solubilized in variousconcentrations of aqueous trifluoroethanol (TFE) was analyzed bycircular dichroism (FIG. 25). Glucagon was the least helical peptidetested, and had calculated helicity of 10, 15 and 33% in TFE solutionsof 0, 10 and 20%, respectively (Table 29). Under the same experimentalconditions, GLP-1 had enhanced helicity of 14, 29 and 55%, demonstratingthat these two peptides differ in primary as well as secondarystructure. There were no significant changes in the helicity of PeptidesX and Y when pegylated, despite the fact that the pegylated portionsrepresent more than 90% of the molecule by mass (Table 29). In contrast,the apparent helicity of Peptide Y in phosphate buffer in the absence ofTFE was approximately double that of Peptide X, from 17% to 36%.Consequently, the pegylated forms of these two chimeric peptides(Peptide X-PEG and Peptide Y-PEG) differed appreciably in secondarystructure (FIG. 25) and the differences in biological properties arelikely a function of these secondary structural differences.

TABLE 29 Percent Helicity Peptide 0% TFE 10% TFE 20% TFE Glucagon 10 1533 GLP-1 14 29 55 Peptide X 17 34 60 Peptide 12 31 — X-PEG Peptide Y 3635 64 Peptide 37 51 — Y-PEG

Example 44

The two peptides (Peptides X and Y) and their 40-kd pegylatedderivatives (Peptides X-PEG and Y-PEG) were assessed for their abilityto stimulate cAMP synthesis in cell-based CRE-luciferase reporter assays(FIG. 26). As shown in Table 30, native glucagon activated glucagonreceptors half-maximally at an effective concentration (EC50) of0.055±0.014 nM and the GLP-1 receptor (GLP-1R) at a much higherconcentration, EC50 of 3.29±0.39 nM. In contrast, GLP-1 activated itsreceptor with an EC50 of 0.028±0.009 nM and proved highly specific inthat interaction at the glucagon receptor (GcgR) occurred at an EC50exceeding 1 μM. The dynamic range in specificity exhibited for thenative ligands at their receptors is in excess of a million. The potencyof Peptide X-PEG at GLP-1R was twice that of native GLP-1 and even moreenhanced at GcgR in a relative sense. However, the GcgR activity wasonly approximately 10% that of native glucagon. The introduction of thelactam restored full glucagon agonism without a change at GLP-1R.Consequently, Peptide Y-PEG is a fully potent, nearly balancedco-agonist relative to the native ligands at the two respectivereceptors. PEGylation of each peptide reduced potency by as much asten-fold at GcgR and five-fold at GLP-1R. The slightly enhanced loss inactivity at GcgR may be a function of the greater relative importance ofthe C-terminal sequence to glucagon receptor interaction. The pegylatedpeptides (Peptides X-PEG and Y-PEG) were slightly less potent at GLP-1Rthan native GLP-1 but still had a subnanomolar EC50. Peptide X-PEG isseven-fold more selective than the lactam version of this peptide, i.e.,Peptide Y-PEG, at the GLP-1R. Therefore, these two DPP-4-resistantpeptides are suitable for sustained in vivo time-action experiments andwell-matched for GLP-1R agonism, but differ in glucagon agonism.

TABLE 30 Glucagon Receptor GLP-1 Receptor EC50 Standard % activity ofEC50 Standard % activity of Peptide (nM) Deviation native glucagon (nM)Deviation native GLP-1 Selectivity* Glucagon 0.055 0.014 100.00 3.2930.389 0.86 0.009 GLP-1 >1000 — <0.008 0.028 0.009 100.00 >12500 PeptideX 0.585 0.125 9.38 0.014 0.002 202.12 21.5 Peptide X-PEG 2.895 0.9631.90 0.036 0.014 78.41 41.4 Peptide Y 0.055 0.011 99.51 0.013 0.005219.78 2.21 Peptide Y-PEG 0.667 0.264 8.22 0.059 0.029 47.61 5.79

Example 45

The 40-kd pegylated peptides Peptides X-PEG and Y-PEG were used assingle weekly subcutaneous (s.c) injections in diet-induced obese (D10)C57B6 mice. A single injection of 325 nmol/kg of Peptide Y-PEG decreasedbody weight over one week by 25.8%, from 50.9±1.4 g to 37.8±0.8 g(p<0.0001, n=8/group). Comparable administration of Peptide X-PEG waseffective but considerably less potent, as the decrease in body weightwas 9% (49.1±1.51 g to 44.68±1.38 g). Saline-injected control mice didnot change their body weight (before: 50.61±1.32 g, after: 50.87±1.46 g;FIG. 27A). The body weight changes were a result of a decrease in fatmass (41.9% for the lactam peptide, 22.2% for open form, 2.3% forcontrols, p<0.001; FIG. 27B) and were paralleled by a significantdecrease in average daily food intake (Peptide Y-PEG: 0.40±0.29 g/day,Peptide X-PEG: 1.83±0.81 g/day, saline: 2.70±0.78 g/day, p<0.0001, FIG.27C). Blood glucose was significantly decreased for both peptides whencompared to control, and slightly more so in Peptide Y-PEG (PeptideY-PEG: −90.1 mg/dL, Peptide X-PEG: −79.6 mg/dL, control: −23.9 g,/dL,p=0.0433; FIG. 27D). The relative difference between the two peptides(Peptide X-PEG and Peptide Y-PEG) was not statistically significant.

Example 46

In a separate experiment, single s.c. injections of six different doses(0, 7, 14, 35, 70, 140 and 350 nmol/kg) of Peptide Y-PEG and PeptideX-PEG demonstrated linearly responsive, dose-dependent decreases in bodyweight and blood glucose (FIGS. 28A, 28B, 28C and 28D). This suggeststhat the observed effects are pharmacologically relevant with noapparent toxicity, other than the indirect effects of rapid, excessiveloss in body weight. The magnitude of the effect was more prominent withPeptide Y-PEG and indicates that the additional element of glucagonagonism improves the potency of the peptide.

Example 47

In a separate experiment, weekly s.c. injections of 70 nmol/kg ofPeptide Y-PEG or Peptide X-PEG decreased body weight of DIO mice by28.1% and 20.1%, respectively (p<0.0001, n=7-8/group; FIG. 29A). Thebody weight changes were associated with a decrease in fat mass (−62.9%for Peptide Y-PEG, −52.2% for Peptide X-PEG, and 5.1% for controls,p<0.0001; FIG. 29B). Long-term effects of these lower doses on foodintake (p=0.95; FIG. 29C) were less impressive than short-term effectswith a higher dose (FIG. 27C). Energy expenditure was increased withPeptide Y-PEG (14.60±0.69 kcal/[kg*h]) and Peptide X-PEG (17.19±1.49kcal/[kg*h]) compared to vehicle (12.71±0.45 kcal/[kg*h]), p=0.0187),whereas the respiratory quotient tended to be decreased (FIGS. 29D and29E; 0.719±0.01 for Peptide Y-PEG, 0.725±0.01 for Peptide X-PEG, and0.755±0.01 for vehicle, p=0.1028), indicating that increasedthermogenesis and altered nutrient partitioning may explain the overallnegative energy balance. Increased energy expenditure was not associatedwith a change in spontaneous physical activity induced thermogenesis(NEAT) since locomotor activity did not differ between treatment groupsand controls (p=0.4281; FIG. 29F). Neither automated online monitoringof acute feeding nor chronic monitoring of food intake revealed anydifferences in caloric intake (automated p=0.667, chronic p=0.9484; FIG.30A).

Blood glucose levels were markedly decreased over the treatment periodstarting at Day 3 after the first injection (mean decrease: PeptideY-PEG −32%, Peptide X-PEG −24.5%, controls: −2.7%, p<0.0001; FIG. 29G).In response to an intraperitoneally (i.p.) glucose challenge on Day 3,blood glucose peaks (FIG. 29H) and profiles (AUC) (FIG. 30F) weremarkedly lower in the two treated groups (Peptide Y-PEG 14183±1072,Peptide X-PEG 13794±824.1) compared to the vehicle-treated controls(34125±3142, p<0.0001). After one month of treatment with Peptide Y-PEGor Peptide X-PEG, plasma insulin was lower in the treatment groups (1194pg/ml, 1034 pg/ml, p=0.0244) compared to controls (2675 pg/ml),suggesting improved insulin sensitivity (FIG. 29I). Plasma C— peptidelevels tended to be decreased after one month of treatment with PeptideY-PEG or Peptide X-PEG (738.8 pg/ml, 624.7 pg/ml) versus vehicle (1077pg/ml) (p=0.108) (FIG. 30G).

To determine if the principal phenomenon generalizes across species,both compounds were administered to diet-induced obese rats (mean weight777.4+/−2.1 g, dose 70 nmol/kg/week, once-a-week injection, 3-weektreatment). Peptide Y-PEG and Peptide X-PEG each decreased body weight(Peptide X-PEG: −11.15+/−0.88%; Peptide Y-PEG: −20.58+/−2.26%, vehicle:1.09+/−0.56%) (p<0.0001) and fat mass of the DIO rats (Peptide X-PEG:−19.17+/−2.03%; Peptide Y-PEG: −33.76+/−4.76%, vehicle: 0.65+/−1.20%;p<0.0001), confirming a species-independent applicability of thisanti-obesity treatment approach.

Example 48

Chronic s.c. treatment over 27 days with Peptide X-PEG and Peptide Y-PEGdecreased total cholesterol in DIO mice (106.9±6.3 mg/dL and 200.8±29.58mg/dL, respectively) relative to vehicle (254.0 25.33 mg/dL, p=0.0441;FIG. 31A). In a separate experiment, DIO mice received 70 nmol/kg s.c.of Peptide X-PEG, Peptide Y-PEG or vehicle on Days 0 and 7 and wereevaluated on Day 9. Peptide Y-PEG decreased plasma triglycerides, LDLcholesterol and total cholesterol (total cholesterol 63.0 2.49 mg/dLcompared to vehicle 177.7±11.8 mg/dL) (p<0.0001), while potentiallycausing a switch from LDL to HDL cholesterol (FIG. 31B). Peptide X-PEGdecreased both LDL and HDL cholesterol but had no significant effect ontriglycerides (FIG. 31C). There was a significant decrease in leptin(3343±723.3 pg/ml for Peptide Y-PEG; 7308±2927 for Peptide X-PEG, and18,642±6124 for vehicle; p=0.0426; FIG. 31D, 31E, 31F). Chronictreatment for 27 days also normalized liver lipid content while controlDIO mice maintained significant liver steatosis (data not shown).

Example 49

One month treatment with Peptide X-PEG or Peptide Y-PEG resulted inincreased phosphorylation of hormone sensitive lipase (HSL) in whiteadipose tissue (WAT) of DIO mice (Peptide X-PEG: 1.135±0.315; PeptideY-PEG: 1.625±0.149; vehicle: 0.597±0.204; p=0.0369; FIG. 32B), implyinga glucagon-specific direct effect on WAT lipolysis. Concomitant with thedecrease in fat mass of mice treated for two weeks at a dose of 35nmol/kg/week with the Peptide Y-PEG and Peptide X-PEG, there was asignificant reduction of adipocyte size in epididymal adipose tissueswhen compared to control mice (data not shown). However, despite havingdecreased fat mass and smaller adipocytes, this short term treatment oftwo weeks with the Peptide Y-PEG and Peptide X-PEG was not associatedwith a significant reduction of adipose tissue macrophage content asquantified by real-time RT-PCR for CD68 (FIG. 33C). Uncoupling protein I(UCP1) levels in brown adipose tissue (BAT) were increased by PeptideX-PEG, but not by Peptide Y-PEG treatment (Peptide X-PEG 2.167±0.429,Peptide Y-PEG 1.287±0.1558, and vehicle 1.0±0.118; p=0.0264; FIG. 32A),consistent with a GLP-1-specific action on BAT resting thermogenesis.Hepatic gene expression reflective of hepatic gluconeogenesis was notaffected by either Peptide X-PEG or Peptide Y-PEG (FIG. 30H and 30I).Histology indicated that pancreatic islets tended to be smallerfollowing Peptide X-PEG treatment (data not shown).

Example 50

In order to dissect the contributions of the GLP-1R and the GcgR agonistcomponents of Peptides X-PEG and Y-PEG, each was administered for onemonth to GLP-1 receptor knock out (GLP-1R −/−) mice maintained onhigh-fat diet. Peptide X-PEG caused a reduction of body weight (p>0.05;FIGS. 34A and 34B) and fat mass (p>0.05; FIG. 34C) compared to saline.Peptide Y-PEG caused a significant decrease in body weight (p=0.0025)and fat mass (p=0.0025) in the GLP-1R −/− mice (FIGS. 34A-34C). PeptideX-PEG had no effect on food intake in GLP-1R −/− mice, while PeptideY-PEG suppressed food intake significantly (p=0.017) (FIG. 34D). PeptideY-PEG (but not Peptide X-PEG) had a tendency to increase blood glucosein a glucose tolerance test in the absence of a functional GLP-1R(p=0.03) (FIGS. 34E and 34F), implying that the GLP-1 component of theco-agonist is needed to protect against glucagon-induced hyperglycemia.

Example 51

As an independent assessment of the effect of Peptides X-PEG and Y-PEGthat can be attributable to glucagon agonism, two additional peptideagonists with comparable GLP-1R potency but markedly different GcgRactivity were studied. The two peptides (Peptides U and V) are relatedto the Peptides X-PEG and Y-PEG. Peptides U and V comprised the aminoacid sequence of SEQ ID NO: 1 with the following modifications: Glu atposition 16, Gln at position 17, Ala at position 18, Lys at position 20,Glu at position 21, Ile at position 23, and Cys at position 24, butcomprised a 20-kd pegylation at the Cys at position 24 and did notcomprise AIB at position 2. Peptide V additionally comprised asubstitution of Gln3 with Glu which selectively reduced glucagon agonismby more than ten-fold. Neither Peptide U nor Peptide V comprised alactam bridge. Treatment of DIO mice each day for one week at 50 nmol/kgs.c. with Peptide V revealed a reduced effect on body weight loweringrelative to the Peptide U (−9.09±0.80 vs. −13.71±0.92 g, respectively(p<0.0001; FIG. 35A).

Example 52

Glucagon peptides comprising a C16 fatty acyl group attached to a Lysresidue via a γ-Glu spacer or a γ-Glu-γ-Glu dipeptide spacer, whereinthe Lys residue is located at position 10 or at the C-terminus (atposition 29), were made as essentially described herein. The peptideswere tested for in vitro activities at the glucagon and GLP-1 receptorsas described herein. The results are shown in Table 31.

TABLE 31 EC50 Peptide SEQ (nM) at EC50 at Acylated ID Glucagon GLP-1 AASpacer NO: Receptor Receptor Chi-2, d-Ser2 Lys10 γE 643 0.011 0.0014Chi-2, d-Ser2 Lys10 γE-γE 644 0.008 0.003 Chi-2, AIB2 Lys10 γE 645 0.0250.0014 Chi-2, AIB2 Lys10 γE-γE 646 0.014 0.0018 Chi-2, AIB2, E3 Lys10None 647 46.084 0.005 Chi-2, AIB2, E3 Lys10 γE-γE 648 2.922 0.004 Chi-2,AIB2, I7 Lys10 γE 649 0.014 0.024 (0.044*) Chi-2, AIB2, I7 Lys10 γE-γE650 0.007 0.010 DMIA1, E16/K20 Lys10 γE 651 0.019 0.006 lactam DMIA1,E16/K20 Lys10 γE-γE 652 0.014 0.004 lactam DMIA1, E16/K20 Lys29 γE 6530.107 0.075 lactam DMIA1, E16/K20 Lys29 γE-γE 654 0.025 0.070 lactamAIB2, AIB16, A18, Lys10 γE-γE 655 0.003 0.004 D28 AIB2, AIB16, A18,Lys10 γE 656 0.006 0.004 D28

Example 53

The peptides shown in Table 32 were made as essentially describedherein:

TABLE 32 SEQ ID Peptide Name NO: Sequence Chimera-2 Aib2C24Mal40KPEG 642H(Aib)QGTFTSDYSKYLDEQAAKEFICWLMNT-amide Chimera-2 625H(Aib)QGTFTSDYSKYLDEQAAKEFICWLMNT-amide Aib2E16K20lactamC24Mal40KPEGGlucagon 626 H(Aib)QGTFTSDYSKYLDERRAKDFVCWLMNT-amideAib2E16K20lactamC24amideMal40KPEG lactam Glucagon 628(Dmia)SQGTFTSDYSKYLDERRAKDFVCWLMNT-amide Dmia1E16K20lactamC24Mal40KPEGlactam Glucagon 629 (Dmia)SQGTFTSDYSKYLDERRAKDFVCWLMNT-OHDmia1E16K20lactamC24Mal40KPEG lactam Glucagon 630(Dmia)SQGTFTSDYSKYLDERRAKDFVCWLMNT-amideDmia1E16K20lactamC24thioether40KPEG lactamChimera2 Aib2E3C24-Thioether40K PEG 631H(Aib)EGTFTSDYSKYLDEQAAKEFICWLMNT-amideGlucagon DMIA1, E3, E15, E16, K20, 632(Dmia)SEGTFTSDYSKYLEERRAKDFVC(PEG40K)WLMNT- C24-Peg amideGlucagon Aib2Aib16C24K10(rErE- 633 H(Aib)QGTFTSDK(rErE-C14)C24PEG40K TE)amide C14)SKYLDAibRRAQDFVC(PEG40K TE)WLMNT-amideGlucagon Aib2Aib16K10(AA- 634 H(Aib)QGTFTSDK(AA- C14)C24PEG40K TE amideC14)SKYLDAibRRAQDFVC(PEG40K TE)WLMNT-amideGlucagon Aib2Aib16K10(AA-C16) amide 635 H(Aib)QGTFTSDK(AA-C16)SKYLDAibRRAQDFVQWLMNT amide Glucagon Aib2Aibl6K10(rErE-C16) 636H(Aib)QGTFTSD K(rErE- amide C16)SKYLDAibRRAQDFVQWLMNT amide

All peptides of Table 32 demonstrated potent in vitro activities at boththe glucagon and GLP-1 receptors, except for the peptides of SEQ ID NOs:624, 631, and 632

Peptides of Set A comprising the amino acid sequence of native glucagon(SEQ ID NO: 1) except for the changes outlined in Table 33 are made asessentially described herein.

TABLE 33 C- DPP-IV Back- Terminal Protection Alpha Helix StabilizationPosition 3 bone* Amide? DMIA at AIB at position 16 Gln (wild- Wild-typeyes position 1 type) AIB at AIB at position 16 Gln (wild- Wild-type yesposition 2 type) d-Ser at AIB at position 16 Gln (wild- Wild-type yesposition 2 type) DMIA at AIB at position 16 Glu Wild-type yes position 1AIB at AIB at position 16 Glu Wild-type yes position 2 d-Ser at AIB atposition 16 Glu Wild-type yes position 2 DMIA at AIB at positions 16 andGln (wild- Wild-type yes position 1 20 type) AIB at AIB at positions 16and Gln (wild- Wild-type yes position 2 20 type) d-Ser at AIB atpositions 16 and Gln (wild- Wild-type yes position 2 20 type) DMIA atAIB at positions 16 and Glu Wild-type yes position 1 20 AIB at AIB atpositions 16 and Glu Wild-type yes position 2 20 d-Ser at AIB atpositions 16 and Glu Wild-type yes position 2 20 DMIA at Glu at position16 and Gln (wild- Wild-type yes position 1 Lys at position 20 type) AIBat Glu at position 16 and Gln (wild- Wild-type yes position 2 Lys atposition 20 type) d-Ser at Glu at position 16 and Gln (wild- Wild-typeyes position 2 Lys at position 20 type) DMIA at Lactam bridge betweenGln (wild- Wild-type yes position 1 side chains of Glu at type) position16 and Lys at position 20 AIB at Lactam bridge between Gln (wild-Wild-type yes position 2 side chains of Glu at type) position 16 and Lysat position 20 d-Ser at Lactam bridge between Gln (wild- Wild-type yesposition 2 side chains of Glu at type) position 16 and Lys at position20 DMIA at Glu at position 16 and Glu Wild-type yes position 1 Lys atposition 20 AIB at Glu at position 16 and Glu Wild-type yes position 2Lys at position 20 d-Ser at Glu at position 16 and Glu Wild-type yesposition 2 Lys at position 20 DMIA at Lactam bridge between GluWild-type yes position 1 side chains of Glu at position 16 and Lys atposition 20 AIB at Lactam bridge between Glu Wild-type yes position 2side chains of Glu at position 16 and Lys at position 20 d-Ser at Lactambridge between Glu Wild-type yes position 2 side chains of Glu atposition 16 and Lys at position 20 DMIA at Glu at position 16 and Gln(wild- Chimera 2 yes position 1 Lys at position 20 type) AIB at Glu atposition 16 and Gln (wild- Chimera 2 yes position 2 Lys at position 20type) d-Ser at Glu at position 16 and Gln (wild- Chimera 2 yes position2 Lys at position 20 type) DMIA at Lactam bridge between Gln (wild-Chimera 2 yes position 1 side chains of Glu at type) position 16 and Lysat position 20 AIB at Lactam bridge between Gln (wild- Chimera 2 yesposition 2 side chains of Glu at type) position 16 and Lys at position20 d-Ser at Lactam bridge between Gln (wild- Chimera 2 yes position 2side chains of Glu at type) position 16 and Lys at position 20 DMIA atGlu at position 16 and Glu Chimera 2 yes position 1 Lys at position 20AIB at Glu at position 16 and Glu Chimera 2 yes position 2 Lys atposition 20 d-Ser at Glu at position 16 and Glu Chimera 2 yes position 2Lys at position 20 DMIA at Lactam bridge between Glu Chimera 2 yesposition 1 side chains of Glu at position 16 and Lys at position 20 AIBat Lactam bridge between Glu Chimera 2 yes position 2 side chains of Gluat position 16 and Lys at position 20 d-Ser at Lactam bridge between GluChimera 2 yes position 2 side chains of Glu at position 16 and Lys atposition 20 *indicates amino acids at positions 17, 28, 21, and 23 aswild-type or as Chimera 2 (Gln at position 17, Ala at position 18, Gluat position 21, and Ile at position 23).

Peptides having the same structure as the peptides of Set A, except thatthe Met at position 27 is replaced with a Norleucine, are made asessentially described herein. These modified peptides are the peptidesof Set B.

Peptides having the same structure as the peptides of Sets A and B,except that the Gln at position 24 is replaced with a Cys covalentlyattached to a 40 kDa PEG, are made as essentially described herein.These pegylated peptides form the peptides of Set C.

Peptides having the same structure as the peptides of Set A, B, or C,except that the Tyr at position 10 is replaced with a Lys covalentlyattached to a C8, C12, C14, C16, or C18 fatty acyl group, are made asessentially described herein. The peptides acylated with a C8 fatty acylgroup form the peptides of Set D. The peptides acylated with a C12 fattyacyl group form the peptides of Set E. The peptides acylated with a C14fatty acyl group form the peptides of Set F. The peptides acylated witha C16 fatty acyl group form the peptides of Set G. The peptides acylatedwith a C18 fatty acyl group form the peptides of Set H.

Peptides having the same structure as the peptides of Sets D through H,except that the fatty acyl group is attached to the Lys at position 10via a spacer, are made as essentially described herein. The peptidescomprising a γ-Glu- γ-Glu spacer form the peptides of Set I. Thepeptides comprising a γ-Glu spacer form the peptides of Set J. Thepeptides comprising an Ala-Ala spacer form the peptides of Set K. Thepeptides comprising a β-Ala- β-Ala spacer form the peptides of Set L.

Example 54

Glucagon peptides comprising SEQ ID NO: 1 with up to 15 amino acidmodifications, a C-terminal extension of 1-21 amino acids, wherein (a)the extension is acylated or alkylated, and/or (b) the extensioncomprises 1-6 positive-charged amino acids, were made as essentiallydescribed herein. The peptides were tested for in vitro activity at theglucagon, GLP-1, and GIP receptors as essentially described in Example14. The structures of the peptides and their in vitro activities aresummarized in Table 34.

TABLE 34 Glucagon Receptor GLP-1 Receptor GIP Receptor SEQ EC50 Standard% relative EC50 Standard % relative EC50 Standard % relative Peptide IDNO: (nM) deviation activity* (nM) deviation activity* (nM) deviationactivity* mt-345 657 0.0183 0.0353 192.90 0.0036 0.0055 152.78 0.05410.0027 4.99 mt-346 658 0.9535 0.0353 3.70 0.0015 0.0055 366.67 0.79610.0027 0.34 mt-347 659 0.0317 0.0353 111.36 0.0082 0.0055 67.07 0.38300.0027 0.70 mt-348 660 0.4327 0.0353 8.16 0.0037 0.0055 148.65 1.76050.0027 0.15 mt-349 661 0.5494 0.0210 3.82 0.0022 0.0063 286.36 1.99060.0036 0.18 mt-350 662 nd nd Nd 0.0107 0.0063 58.88 77.7229 0.0036 0.00mt-351 663 0.1963 0.0210 10.70 0.0042 0.0063 150.00 4.1217 0.0036 0.09mt-352 664 nd nd nd 0.0273 0.0063 23.08 51.1125 0.0036 0.01 mt-361 6661.7701 0.0639 3.61 0.0109 0.0247 226.35 1.0488 0.0031 0.29 mt-362 6670.0768 0.0639 83.17 0.102  0.0247 240.96 0.0782 0.0031 3.95 mt-363 6680.8230 0.0639 7.76 0.0037 0.0247 671.66 0.4250 0.0031 0.73 mt-364 6690.0183 0.0639 349.97 0.0070 0.0247 354.68 0.0197 0.0031 15.66 *Activityrelative to the native hormone at the indicated receptor

As shown in Table 34, all unpegylated peptides demonstrated agonistactivity at the GIP receptor that was greater than 0.1%, Peptidesmt-345, mt-362, and mt-364 demonstrated exceptionally potent activity atthe GIP receptor. Peptides mt-350, mt-351, and mt-352 demonstratedactivities less than 0.1% likely due to the fact that these peptideswere pegylated.

Example 55

The in vivo effects of mt-345 (having the structure of SEQ ID NO: 657)were compared to the effects of other acylated peptides (Liraglutide,mt-261 (SEQ ID NO: 670), and mt-347 (SEQ ID NO: 659)) and a non-acylatedpeptide (mt-348; SEQ ID NO: 660). Peptides mt-345, mt-261, and mt-347comprised the amino acid sequence of glucagon (SEQ ID NO: 1) with aminoacid modifications and further comprised an extension of GPSSGAPPPS (SEQID NO: 26) C-terminal to the amino acid at position 29 followed by anacylated Lys as position 40. Peptide mt-345 comprised AIB at position 2,Glu at position 16, Gln at position 17, Ala at position 18, Lys atposition 20, Glu at position 21, Ile at position 23, Cys at position 24,Gly at position 29, SEQ ID NO: 26 c-terminal to the Gly at position 29,and an acylated Lys as position 40. The acyl group of mt-345 was a C14fatty acyl group. Peptide mt-261 had a similar structure to that ofmt-345, except that mt-261 comprised a Tyr at position 1, Glu atposition 3, Ile at position 12, Lys at position 16, AIB at position 20,Val at position 23, Asn at position 24, Leu at position 27, Ala atposition 28, and a C16 fatty acyl group at position 40. The structuresof mt-347 and mt-348 were identical to the structure of mt-345, exceptthat mt-347 and mt-348 comprised an AIB at position 16. The structure ofmt-347 was the same as that of mt-348 except that mt-347 comprised a C14fatty acyl group at position 40, whereas mt-348 lacked an acyl group.

The in vitro activities at the glucagon, GLP-1, and GIP receptors of thepeptides were tested as essentially described in Example 14. Therelative activities are shown in Table 35.

TABLE 35 % Relative activity at the Receptor for Peptide Glucagon GLP-1GIP Liraglutide 0.04 138 n/a mt-261 13.4 372 700 mt-345 193 153 5 mt-347111 67 0.7 mt-348 8.2 149 0.2 % relative activity is relative to thenative hormone of the indicated receptor

The peptides were administered daily for two weeks to 11 groups of 8C57Bl/6 mice by subcutaneous injection at a dose of 10 or 30 nmol/kg.The initial average body weight of the mice was 51 g. The mice were 6months old and had been on a diabetogenic diet for 4 months. One groupof mice received a vehicle control and another received a glucagon amidecontrol peptide which comprised the amino acid sequence of SEQ ID NO: 1with a C-terminal amidation in place of the C-terminal alphacarboxylate.

The body weight of each group of mice receiving a peptide steadilydeclined over the course of the study, as compared to the vehiclecontrol. However, as shown in FIG. 36, the total change in body weightof mice receiving 10 nmol/kg of mt-261 or mt-345 was the mostsignificant (around 20%). Mice that were administered the glucagon amidepeptide also demonstrated a similar total decrease in body weight asthose of mt-261 and mt-345, although the dose of the glucagon amide wasthree times as much as the dose of mt-261 and mt-345. The effects onbody weight were clearly more significant than the effects ofliraglutide.

Food intake by the groups of mice were also evaluated during the study.Mice that received the mt-261 or mt-345 peptide demonstrated the lowestfood intake.

Blood glucose levels of the mice were also studied. Mice that wereadministered liraglutide, mt-261, or mt-345 demonstrated a totaldecrease in blood glucose levels on Day 7 (7 days after the firstadministration of peptide). The extent of the decrease was less howeverwhen measured on Day 14.

Example 56

Glucagon peptides comprising an acylated C-terminal extension were madeas essentially described herein. Peptides mt-278, mt-261, mt-297, andmt-358 comprised the amino acid sequence of SEQ ID NO: 1 with thefollowing amino acid modifications: Tyr at position 1, AIB at positions2 and 20, Lys at position 16, Ile at position 12, Gln at position 17,Ala at position 18, Glu at position 21, Gly at position 29, the aminoacid sequence of SEQ ID NO: 26 C-terminal to the amino acid at position29, and an acylated Lys at position 40. Peptide mt-364 varied instructure from the other peptides by comprising a His at position 1, Gluat position 16 and a Lys at position 20. The full descriptions of thestructures of these peptides are described in the sequence listingattached hereto: mt-364 (SEQ ID NO: 669), mt-261 (SEQ ID NO: 270),mt-278 (SEQ ID NO: 271), mt-297 (SEQ ID NO: 272), and mt-358 (SEQ ID NO:673).

The peptides were tested for in vitro activities at the glucagon, GLP-1,and GIP receptors as essentially described in Example 14. The activitiesof the peptides in comparison to the native hormones at the indicatedreceptor are shown in Table 36.

TABLE 36 % Relative activity at the Receptor for Peptide Glucagon GLP-1GIP mt-261 31.62 299.24 297.6 mt-278 761.36 319.58 266.40 mt-297 276.50144.16 68.63 mt-358 710.29 11.89 313.61 mt-364 349.97 354.68 15.66 %relative activity is relative to the native hormone of the indicatedreceptor

The peptides were then tested in vivo for their effects on body weight,food intake, blood glucose levels, and fat mass in eight-month oldC57B1/6 mice that had been on a diabetogenic diet for 6 months. Elevengroups of eight mice were subcutaneously injected with 10 nmol/kg ofpeptide daily for one week. The mice had an initial body weight of 53 g.

The body weight of mice that were injected with mt-278, mt-261, ormt-364 steadily decreased over the course of the study, as compared tomice that were injected with vehicle only. As shown in FIG. 37, miceinjected with mt-364 demonstrated the greatest decrease in body weightby the end of the week-long study, achieving a weight loss of greaterthan 15%. Mice injected with mt-278 or mt-261 also demonstratedsubstantial (greater than 10%) decreases in body weight.

Total food intake by the mice was monitored and by the end of the study,total food intake by mice injected with mt-364 or mt-261 demonstrated atotal food intake which was less than 50% of the total food intake bymice injected with vehicle only.

The fat mass of mice was measured throughout the study. Mice injectedwith mt-364, mt-278, or mt-261 exhibited a fat mass which was less thanthe fat mass of mice injected with vehicle only.

The effects on blood glucose levels were additionally evaluated. On thelast day of the study (Day 7), mice injected with mt-364 or mt-261exhibited a decrease in blood glucose levels that was greater than 60%as compared to the blood glucose levels measured on Day 0. Mice injectedwith mt-278 additionally demonstrated a substantial decrease in bloodglucose (about 40% decrease).

Example 57

Analogs of glucagon having a C-terminal amide in place of the C-terminalalpha carboxylate were made as essentially described herein:

Peptide 83 comprised the structure of SEQ ID NO: 1 with AIB at position2, AIB at position 16, and Lys at position 10, which Lys comprised a C16fatty acyl group via a γ-Glu-γ-Glu dipeptide spacer.

Peptide 900 comprised the structure of SEQ ID NO: 1 with AIB at position2, AIB at position 16, a C-terminal extension comprising SEQ ID NO: 26,and Lys at position 40, which Lys comprised a C16 fatty acyl group via aγ-Glu-γ-Glu dipeptide spacer.

Peptide 901 comprised the structure of SEQ ID NO: 1 with AIB at position2, AIB at position 16, a C-terminal extension comprising SEQ ID NO: 26,and Lys at position 10, which Lys comprised a C16 fatty acyl group via aγ-Glu-γ-Glu dipeptide spacer.

Peptide mt-364 comprised the structure of SEQ ID NO: 669.

The analogs were tested for in vitro activity at each of the glucagon,GLP-1, and GIP receptors as essentially described herein. The resultsare shown in Table 37.

TABLE 37 % Relative activity at the Receptor for Peptide Glucagon GLP-1GIP  83 211 84 nd 900 560 383 2.89 901 560 460 8.65 mt-364 349.97 354.6815.66 nd = not determined

Nine groups of 8 DIO mice (strain: C57B16 WT) were subcutaneouslyinjected daily for 7 days with 10 nmol/kg of one of the peptides ofTable 37. The average initial body weight of the mice was 57.6 g. Themice were approximately 10 months old and had been on a high fat dietfor about 8 months.

The total change in body weight was measured on Day 7. As shown in FIG.38, all mice injected with a peptide of Table 37 demonstrated a decreasein body weight as compared to vehicle control. Among Peptides 83, 900and 901, all of which have a similar peptide structure, Peptides 900 and901 demonstrated the greatest decreases in body weight. Interestingly,each of Peptides 900 and 901 comprised a C-terminal extension and wasacylated, in contrast to Peptide 83, which was acylated but did notcomprise the C-terminal extension. Peptide 901 which was acylated atposition 10 demonstrated a greater decrease (˜15% decrease) than Peptide900 (˜10% decrease) which was acylated at position 40. The in vivoeffects on body weight of these peptides, however, were still not assignificant as those of MT-364. Mice that were injected with MT-364demonstrated about a 20% decrease.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An analog of a glucagon peptide, the analog comprising: (i) SEQ IDNO: 1 with at least one and up to 10 amino acid modifications, whereinat least one of the amino acid modifications confers a stabilized alphahelix structure in the C-terminal portion of the analog; (ii) anextension of 1 to 21 amino acids C-terminal to the amino acid atposition 29, and (iii) at least one of the following: (a) at least oneof the amino acids of the extension located at any of positions 37-43(according to the numbering of SEQ ID NO: 1) comprises an acyl or alkylgroup which is non-native to a naturally-occurring amino acid, (b) 1-6amino acids of the extension are positive-charged amino acids, (c) theanalog comprises an amino acid comprising an acyl or alkyl group, whichis non-native to a naturally-occurring amino acid, at position 10 of theanalog, or (d) a combination of (a), (b), and (c); wherein, when theanalog lacks a hydrophilic moiety, the analog exhibits at least 0.1%activity of native GIP at the GIP receptor.
 2. (canceled)
 3. (canceled)4. The analog of claim 1, further comprising one or more of: Gln atposition 17, Ala at position 18, Glu at position 21, Ile at position 23,and Ala or Cys at position 24, or one or more conservative amino acidsubstitutions thereof.
 5. The analog of claim 4, comprising a C-terminalamide.
 6. The analog of claim 1 comprising an amino acid substitution atposition 1, position 2, or positions 1 and 2, wherein the amino acidsubstitution(s) achieve DPP-IV protease resistance.
 7. (canceled)
 8. Theanalog of claim 6, wherein the Ser at position 2 is substituted with anamino acid selected from the group consisting of: D-serine, alanine,D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB).
 9. The analog of claim 8, wherein the amino acidat position 1 comprises an imidazole ring.
 10. The analog of claim 9,wherein the amino acid at position 1 is His.
 11. (canceled) 12.(canceled)
 13. The analog of claim 9, comprising amino acidmodifications at one, two or all of positions 27, 28 and
 29. 14. Theanalog of claim 13, wherein (a) the Met at position 27 is substitutedwith a large aliphatic amino acid, optionally Leu, (b) the Asn atposition 28 is substituted with a small aliphatic amino acid, optionallyAla, (c) the Thr at position 29 is substituted with a small aliphaticamino acid, optionally Gly, or (d) a combination of two or all of (a),(b), and (c).
 15. (canceled)
 16. The analog of claim 1, comprising oneor more of the following modifications: a. Ser at position 2 substitutedwith Ala; b. Gln at position 3 substituted with Glu or a glutamineanalog; c. Thr at position 7 substituted with a Ile; d. Tyr at position10 substituted with Trp or an amino acid comprising an acyl or alkylgroup which is non-native to a naturally-occurring amino acid; e. Lys atposition 12 substituted with Ile; f. Asp at position 15 substituted withGlu; g. Ser at position 16 substituted with Glu; h. Gln at position 20substituted with Ser, Thr, Ala, AIB; i. Gln at position 24 substitutedwith Ser, Thr, Ala, AIB; j. Met at position 27 substituted with Leu orNle; k. Asn at position 28 substituted with a charged amino acid,optionally, Asp or Glu; and l. Thr at position 29 substituted with Glyor a charged amino acid, optionally, Asp or Glu.
 17. The analog of claim1, comprising (i) the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 26)or XGPSSGAPPPS (SEQ ID NO: 674), wherein X is any amino acid, (ii) anamino acid sequence which has at least 80% sequence identity to SEQ IDNO: 26 or SEQ ID NO: 674, or (iii) the amino acid sequence of (i) or(ii) with one or more conservative amino acid substitutions, wherein theamino acid sequence is C-terminal to the amino acid at position 29 ofthe analog.
 18. The analog of claim 17, comprising the amino acidsequence of GPSSGAPPPS (SEQ ID NO: 26) or XGPSSGAPPPS (SEQ ID NO: 674),wherein X is any amino acid, C-terminal to the amino acid at position 29of the analog.
 19. The analog of claim 1, wherein the amino acidcomprising an acyl or alkyl group is an amino acid of Formula I, II, orIII.
 20. The analog of claim 19, wherein the amino acid comprising anacyl or alkyl group is Lys.
 21. The analog of claim 1, wherein the aminoacid comprising an acyl or alkyl group is located at any of positions37, 38, 39, 40, 41, 42 or 43 of the analog.
 22. The analog of claim 21,wherein the amino acid comprising an acyl or alkyl group is located atposition 40 of the analog.
 23. The analog of claim 1, wherein the acylgroup is a C4 to C30 fatty acyl group.
 24. The analog of claim 1,wherein the acyl or alkyl group is covalently attached to the side chainof the amino acid via a spacer.
 25. (canceled)
 26. The analog of claim24, wherein the spacer is an amino acid or dipeptide.
 27. (canceled) 28.(canceled)
 29. The analog of claim 24, wherein the total length of thespacer and the acyl group is about 14 to about 28 atoms in length. 30.The analog of claim 23, wherein the acyl group is a C12 to C18 fattyacyl group. 31-50. (canceled)
 51. The analog of claim 1, wherein (i) theGIP potency of the analog is within about 500-fold of GLP-1 potency ofthe analog, (ii) wherein the GIP potency of the analog is within about500-fold of the glucagon potency of the analog, or (iii) both (i) and(ii).
 52. (canceled)
 53. A dimer comprising two glucagon peptides,wherein at least one of the glucagon peptides is an analog of claim 1,bound to one another through a linker.
 54. A pharmaceutical compositioncomprising an analog of claim 1, and a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 55. (canceled)
 56. Amethod of treating hyperglycemia or diabetes, said method comprisingadministering an effective amount of a pharmaceutical composition ofclaim
 54. 57. (canceled)
 58. A method of reducing weight gain orinducing weight loss, said method comprising administering an effectiveamount of a pharmaceutical composition of claim
 54. 59. An analog of aglucagon peptide, the analog comprising: (i) SEQ ID NO: 1 with at leastone and up to 10 amino acid modifications, wherein (i) the analogcomprises an intramolecular bridge between the side chains of an aminoacid at position i and an amino acid at position i+4 or between the sidechains of amino acids at positions j and j+3, wherein i is 12, 13, 16,17, 20 or 24 and j is 17, (ii) one, two, three or more of positions 16,20, 21 or 24 of the analog are substituted with an α,α-disubstitutedamino acid, or (iii) both (i) and (ii); (ii) an extension of 1 to 21amino acids C-terminal to the amino acid at position 29 comprising (i)the amino acid sequence of GPSSGAPPPS (SEQ ID NO: 26) or XGPSSGAPPPS(SEQ ID NO: 674), wherein X is any amino acid, (ii) an amino acidsequence which has at least 80% sequence identity to SEQ ID NO: 26 orSEQ ID NO: 674, or (iii) the amino acid sequence of (i) or (ii) with oneor more conservative amino acid substitutions, wherein the amino acidsequence is C-terminal to the amino acid at position 29 of the analog;and (iii) at least one of the following: (a) at least one of the aminoacids of the extension located at any of positions 37-43 (according tothe numbering of SEQ ID NO: 1) comprises an acyl or alkyl group which isnon-native to a naturally-occurring amino acid, (b) the analog comprisesan amino acid comprising an acyl or alkyl group, which is non-native toa naturally-occurring amino acid, at position 10 of the analog, or (c) acombination thereof; wherein, when the analog lacks a hydrophilicmoiety, the analog exhibits at least 0.1% activity of native GIP at theGIP receptor.
 60. An analog of a glucagon peptide, the analogcomprising: SEQ ID NO: 1 with at least one and up to 10 amino acidmodifications, wherein at least one of the amino acid modificationsconfers a stabilized alpha helix structure in the C-terminal portion ofthe analog; an extension of 1 to 21 amino acids C-terminal to the aminoacid at position 29, and at least one of the following: (a) at least oneof the amino acids of the extension located at any of positions 37-43(according to the numbering of SEQ ID NO: 1) comprises an acyl or alkylgroup which is non-native to a naturally-occurring amino acid, (b) theanalog comprises an amino acid comprising an acyl or alkyl group, whichis non-native to a naturally-occurring amino acid, at position 10 of theanalog, or (c) a combination of (a) and (b); wherein the amino acid atposition 1 comprises an imidazole ring; wherein position 2 is an aminoacid selected from the group consisting of: D-serine, alanine,D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and aminoisobutyric acid (AIB), wherein, when the analog lacks a hydrophilicmoiety, the analog exhibits at least 0.1% activity of native GIP at theGIP receptor.